Psca: prostate stem cell antigen and uses thereof

ABSTRACT

The invention provides a novel prostate cell-surface antigen, designated Prostate Stem Cell Antigen (PSCA), which is widely over-expressed across all stages of prostate cancer, including high grade prostatic intraepithelial neoplasia (PIN), androgen-dependent and androgen-independent prostate tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/297,562, filedJun. 5, 2014, which is a continuation of U.S. Ser. No. 12/169,561, filedJul. 8, 2008, issued as U.S. Pat. No. 8,759,006, which is a continuationof U.S. Ser. No. 10/769,308, filed Jan. 29, 2004, issued as U.S. Pat.No. 7,407,656, which is a continuation of U.S. Ser. No. 10/225,784,filed Aug. 21, 2002, issued as U.S. Pat. No. 7,527,786, which is adivisional of U.S. Ser. No. 09/564,329, filed May 3, 2000, issued asU.S. Pat. No. 6,541,212, which is a continuation-in-part (CIP) of U.S.Ser. No. 09/359,326, filed Jul. 20, 1999, which claims the benefit ofU.S. Ser. No. 60/124,658, filed Mar. 16, 1999, of U.S. Ser. No.60/120,536, filed Feb. 17, 1999, and of U.S. Ser. No. 60/113,230, filedDec. 21, 1998, and which is a CIP of U.S. Ser. No. 09/318,503, filed May25, 1999, issued as U.S. Pat. No. 6,261,791, which is a CIP of U.S. Ser.No. 09/251,835, filed Feb. 17, 1999, issued as U.S. Pat. No. 6,261,789,which is a CIP of U.S. Ser. No. 09/203,939, filed Dec. 2, 1998, issuedas U.S. Pat. No. 6,258,939, which is a CIP of U.S. Ser. No. 09/038,261,filed Mar. 10, 1998, issued as U.S. Pat. No. 6,267,960, which claims thebenefit of U.S. Ser. No. 60/228,816, filed Mar. 10, 1997, of U.S. Ser.No. 60/071,141 filed Jan. 12, 1998, and of U.S. Ser. No. 60/074,675,filed Feb. 13, 1998. U.S. Ser. No. 12/169,561 also is a continuation ofU.S. Ser. No. 10/997,735, filed Nov. 23, 2004, which is a continuationof U.S. Ser. No. 10/225,779, filed Aug. 21, 2002, which is a divisionalof U.S. Ser. No. 09/564,329, filed May 3, 2000, issued as U.S. Pat. No.6,541,212, which is a CIP of U.S. Ser. No. 09/359,326, filed Jul. 20,1999, which claims the benefit of U.S. Ser. No. 60/124,658, filed Mar.16, 1999; of U.S. Ser. No. 60/120,536, filed Feb. 17, 1999; and of U.S.Ser. No. 60/113,230, filed Dec. 21, 1998, and which is a CIP of U.S.Ser. No. 09/318,503, filed May 25, 1999, issued as U.S. Pat. No.6,261,791, which is a CIP of U.S. Ser. No. 09/251,835, filed Feb. 17,1999, issued as U.S. Pat. No. 6,261,789, which is a CIP of U.S. Ser. No.09/203,939, filed Dec. 2, 1998, issued as U.S. Pat. No. 6,258,939, whichis a CIP of U.S. Ser. No. 09/038,261, filed Mar. 10, 1998, issued asU.S. Pat. No. 6,267,960, which claims the benefit of U.S. Ser. No.60/228,816, filed Mar. 10, 1997, of U.S. Ser. No. 60/071,141, filed Jan.12, 1998, and of U.S. Ser. No. 60/074,675, filed Feb. 13, 1998. Thecontents of all of the following applications are incorporated byreference into the present application: U.S. Ser. No. 12/169,561, filedJun. 5, 2014; U.S. Ser. No. 09/359,326, filed Jul. 20, 1999; U.S. Ser.No. 09/318,503, filed May 25, 1999; U.S. Ser. No. 09/251,835, filed Feb.17, 1999; U.S. Ser. No. 09/203,939, filed Dec. 2, 1998; U.S. Ser. No.09/038,261, filed Mar. 10, 1998; U.S. Ser. No. 08/814,279, filed Mar.10, 1997; U.S. Ser. No. 60/017,141, filed Jan. 12, 1998; U.S. Ser. No.60/074,675, filed Feb. 13, 1998; U.S. Ser. No. 60/124,658, filed Mar.16, 1999; U.S. Ser. No. 60/120,536, filed Feb. 17, 1999; and U.S. Ser.No. 60/113,230, filed Dec. 21, 1998.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file SequenceListing_079822-1009605.txt,created on May 26, 2016, 25,854 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer cause the death of well over ahalf-million people each year, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the leading causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients that initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience significant physical debilitations followingtreatment.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markers and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

Recently, there has been a particularly strong interest in identifyingcell surface tumor-specific antigens which might be useful as targetsfor various immunotherapeutic or small molecule treatment strategies. Alarge number of such cell-surface antigens have been reported, and somehave proven to be reliably associated with one or more cancers. Muchattention has been focused on the development of novel therapeuticstrategies which target these antigens. However, few truly effectiveimmunological cancer treatments have resulted.

The use of monoclonal antibodies to tumor-specific or over-expressedantigens in the treatment of solid cancers is instructive. Althoughantibody therapy has been well researched for some 20 years, only veryrecently have corresponding pharmaceuticals materialized. One example isthe humanized anti-HER2/neu monoclonal antibody, Herceptin, recentlyapproved for use in the treatment of metastatic breast cancersoverexpressing the HER2/neu receptor. Another is the human/mousechimeric anti-CD20/B cell lymphoma antibody, Rituxan, approved for thetreatment of non-Hodgkin's lymphoma. Several other antibodies are beingevaluated for the treatment of cancer in clinical trials or inpre-clinical research, including a chimeric and a fully human IgG2monoclonal antibody specific for the epidermal growth factor receptor(Slovin et al., 1997, Proc. Am. Soc. Clin. Oncol. 16:311; Falcey et al.,1997, Proc. Am. Soc. Clin. Oncol. 16:383; Yang et al., 1999, Cancer Res.59: 1236). Evidently, antibody therapy is finally emerging from a longembryonic phase. Nevertheless, there is still a very great need for new,more-specific tumor antigens for the application of antibody and otherbiological therapies. In addition, there is a corresponding need fortumor antigens which may be useful as markers for antibody-baseddiagnostic and imaging methods, hopefully leading to the development ofearlier diagnosis and greater prognostic precision.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis more or less the same for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of this disease,second only to lung cancer. Despite the magnitude of these figures,there is still no effective treatment for metastatic prostate cancer.Surgical prostatectomy, radiation therapy, hormone ablation therapy, andchemotherapy remain as the main treatment modalities. Unfortunately,these treatments are clearly ineffective for many. Moreover, thesetreatments are often associated with significant undesirableconsequences.

On the diagnostic front, the serum PSA assay has been a very usefultool. Nevertheless, the specificity and general utility of PSA is widelyregarded as lacking in several respects. Neither PSA testing, nor anyother test nor biological marker has been proven capable of reliablyidentifying early-stage disease. Similarly, there is no marker availablefor predicting the emergence of the typically fatal metastatic stage ofthe disease. Diagnosis of metastatic prostate cancer is achieved by opensurgical or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy analysis.Clearly, better imaging and other less invasive diagnostic methods offerthe promise of easing the difficulty those procedures place on apatient, as well as improving therapeutic options. However, until thereare prostate tumor markers capable of reliably identifying early-stagedisease, predicting susceptibility to metastasis, and precisely imagingtumors, the management of prostate cancer will continue to be extremelydifficult. Accordingly, more specific molecular tumor markers areclearly needed in the management of prostate cancer.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54: 1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252). PCTA-1, anovel galectin, is largely secreted into the media of expressing cellsand may hold promise as a diagnostic serum marker for prostate cancer(Su et al., 1996). Vaccines for prostate cancer are also being activelyexplored with a variety of antigens, including PSM and PSA.

SUMMARY OF THE INVENTION

The invention provides a novel prostate cell-surface antigen, designatedProstate Stem Cell Antigen (PSCA), which is widely over-expressed acrossall stages of prostate cancer, including high grade prostaticintraepithelial neoplasia (PIN), androgen-dependent andandrogen-independent prostate tumors. The PSCA gene shows 30% homologyto stem cell antigen-2 (SCA-2), a member of the Thy-1/Ly-6 family ofglycosylphosphatidylinositol (GPI)-anchored cell surface antigens, andencodes a 123 amino acid protein with an amino-terminal signal sequence,a carboxy-terminal GPI-anchoring sequence, and multiple N-glycosylationsites. PSCA mRNA expression is highly upregulated in both androgendependent and androgen independent prostate cancer xenografts. In situmRNA analysis localizes PSCA expression to the basal cell epithelium,the putative stem cell compartment of the prostate. Flow cytometricanalysis demonstrates that PSCA is expressed predominantly on the cellsurface and is anchored by a GPI linkage. Fluorescent in situhybridization analysis localizes the PSCA gene to chromosome 8q24.2, aregion of allelic gain in more than 80% of prostate cancers.

PSCA may be an optimal therapeutic target in view of its cell surfacelocation, greatly upregulated expression in certain types of cancer suchas prostate cancer cells. In this regard, the invention providesantibodies capable of binding to PSCA which can be used therapeuticallyto destroy or inhibit the growth of such cancer cells, or to block PSCAactivity. In addition, PSCA proteins and PSCA-encoding nucleic acidmolecules may be used in various immunotherapeutic methods to promoteimmune-mediated destruction or growth inhibition of tumors expressingPSCA.

PSCA also may represent an ideal prostate cancer marker, which can beused to discriminate between malignant prostate cancers, normal prostateglands and non-malignant neoplasias. For example, PSCA is expressed atvery high levels in prostate cancer in relation to benign prostatichyperplasia (BPH). In contrast, the widely used prostate cancer markerPSA is expressed at high levels in both normal prostate and BPH, but atlower levels in prostate cancer, rendering PSA expression useless fordistinguishing malignant prostate cancer from BPH or normal glands.Because PSCA expression is essentially the reverse of PSA expression,analysis of PSCA expression can be employed to distinguish prostatecancer from non-malignant conditions.

The genes encoding both human and murine PSCA have been isolated andtheir coding sequences elucidated and provided herein. Also provided arethe amino acid sequences of both human and murine PSCA. The inventionfurther provides various diagnostic assays for the detection,monitoring, and prognosis of prostate cancer, including nucleicacid-based and immunological assays. PSCA-specific monoclonal andpolyclonal antibodies and immunotherapeutic and other therapeuticmethods of treating prostate cancer are also provided. These and otheraspects of the invention are further described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B. Nucleotide (FIG. 1A) (SEQ ID NO:1) and translated amino acid(FIG. 1B) (SEQ ID NO:2) sequences of a cDNA encoding human PSCA (ATCCDesignation 209612).

FIG. 2. Nucleotide sequence (SEQ ID NO:3) of a cDNA encoding murine PSCAhomologue (SEQ ID NO:4).

FIG. 3. Alignment of amino acid sequences of human PSCA (SEQ ID NO:2),murine PSCA (SEQ ID NO:6), and human stem cell antigen-2 (hSCA-2) (SEQID NO:5). Shaded regions highlight conserved amino acids. Conservedcysteines are indicated by bold lettering. Four predictedN-glycosylation sites in PSCA are indicated by asterisks. The underlinedamino acids at the beginning and end of the protein represent N terminalhydrophobic signal sequences and C terminal GPI-anchoring sequences,respectively.

FIG. 4. Hydrophobicity plot of human PSCA.

FIG. 5. Chou-Fassman analysis of human PSCA.

FIG. 6. A western blot showing that monoclonal antibody 1G8 binds LAPC9(PSCA positive control) and a transitional cell carcinoma (bladdercarcinoma) designated bladder (Rob).

FIG. 7A-B. Restricted Expression of PSCA mRNA in normal and canceroustissues. (FIG. 7A) RT-PCR analysis of PSCA expression in normal humantissues demonstrating high expression in prostate, placenta, andtonsils. 1 ng of reverse-transcribed first strand cDNA (Clontech, PaloAlto, Calif.) from the indicated tissues was amplified with PSCA genespecific primers. Data shown are from 30 cycles of amplification. (FIG.7B) RT-PCR analysis of PSCA expression demonstrating high level inprostate cancer xenografts and normal tissue. 5 ng ofreverse-transcribed cDNA from the indicated tissues were amplified withPSCA gene specific primers. Amplification with β-actin gene specificprimers demonstrate normalization of the first strand cDNA of thevarious samples. Data shown are from 25 cycles of amplification. AD,androgen-dependent; AI, androgen-independent; IT, intratibial xenograft;C.L., cell line.

FIG. 8A-C. Schematic representation of (FIG. 8A) human Thy-1/Ly-6, (FIG.8B) murine PSCA, and (FIG. 8C) human PSCA gene structures.

FIG. 9A-B. Northern blot analysis of PSCA RNA expression. (FIG. 9A)Total RNA from normal prostate and LAPC-4 androgen dependent (AD) andindependent (AI) prostate cancer xenografts were analyzed using PSCA orPSA specific probes. Equivalent RNA loading and RNA integrity weredemonstrated separately by ethidium staining for 18S and 28S RNA. (FIG.9B) Human multiple tissue Northern blot analysis of PSCA RNA. The filterwas obtained from Clontech (Palo Alto, Calif.) and contains 2 ug ofpolyA RNA in each lane.

FIG. 10A-C. Northern blot comparison of (FIG. 10A) PSCA, (FIG. 10B)PSMA, and (FIG. 10C) PSA RNA expression in prostate cancer xenograftsand tumor cell lines. PSCA and PSMA demonstrate high level prostatecancer specific gene expression. 10 μg of total RNA from the indicatedtissues were size fractionated on an agarose/formaldehyde gel,transferred to nitrocellulose, and hybridized sequentially with³²P-labelled probes representing PSCA, PSMA, and PSA cDNA fragments.Shown are 4 hour and 72 hour autoradiographic exposures of the membraneand the ethidium bromide gel demonstrating equivalent loading ofsamples. BPH, benign prostatic hyperplasia; AD, androgen-dependent; AI,androgen-independent; IT, intratibial xenograft; C.L., cell line.

FIG. 11A-C. In situ hybridization with antisense riboprobe for humanPSCA RNA on normal and malignant prostate specimens. (FIG. 11A) PSCA RNAis expressed by a subset of basal cells within the basal cell epithelium(black arrows), but not by the terminally differentiated secretory cellslining the prostatic ducts (400× magnification). (FIG. 11B) PSCA RNA isexpressed strongly by a high grade prostatic intraepithelial neoplasia(PIN) (black arrow) and by invasive prostate cancer glands (yellowarrows), but is not detectable in normal epithelium (green arrow) at 40×magnification. (FIG. 11C) Strong expression of PSCA RNA in a case ofhigh grade carcinoma (200× magnification).

FIG. 12A-C. Biochemical analysis of PSCA protein. (FIG. 12A) PSCAprotein was immunoprecipitated from 293T cells transiently transfectedwith a PSCA construct and then digested with either N-glycosidase F orO-glycosidase, as described in Materials and Methods. (FIG. 12B) PSCAprotein was immunoprecipitated from 293T transfected cells, as well asfrom conditioned media of these cells. Cell-associated PSCA migrateshigher than secreted or shed PSCA on a 15% polyacrylamide gel. (FIG.12C) FACS analysis of mock-transfected 293T cells, PSCA-transfected 293Tcells and LAPC-4 prostate cancer xenograft cells using an affinitypurified polyclonal anti-PSCA antibody. Cells were not permeabilized inorder to detect only surface expression. The y axis represents relativecell number and the x axis represents fluorescent staining intensity ona logarithmic scale. FIG. 13. In situ hybridization of biotin-labeledPSCA probes to human metaphase cells from phytohemagglutinin-stimulatedperipheral blood lymphocytes. The chromosome 8 homologues are identifiedwith arrows; specific labeling was observed at 8q24.2. The inset showspartial karyotypes of two chromosome 8 homologues illustrating specificlabeling at 8q24.2 (arrowheads). Images were obtained using a ZeissAxiophot microscope coupled to a cooled charge coupled device (CCD)camera. Separate images of DAPI stained chromosomes and thehybridization signal were merged using image analysis software (NU200and Image 1.57).

FIG. 14A-C. Flow Cytometric analysis of cell surface PSCA proteinexpression on (FIG. 14A) prostate cancer xenograft (LAPC-9), (FIG. 14B)prostate cancer cell line (LAPC-4) and (FIG. 14C) normal prostateepithelial cells (PreC) using anti-PSCA monoclonal antibodies 1G8(green) and 3E6 (red), mouse anti-PSCA polyclonal serum (blue), orcontrol secondary antibody (black). See Example 5 for details.

FIG. 15A-B. (FIG. 15A) An epitope map for each of the seven disclosedantibodies. (FIG. 15B) Epitope mapping of anti-PSCA monoclonalantibodies conducted by Western blot analysis of GST-PSCA fusionproteins.

FIG. 16A-B. (FIG. 16A) Alignment of amino acid sequences of hSCA-2 (SEQID NO:5), hPSCA (SEQ ID NO:2), and mPSCA (SEQ ID NO:6). (FIG. 16B) Aschematic diagram showing that PSCA is a GPI-anchored protein.

FIG. 17. A photograph showing a FISH analysis of PSCA and c-myc GeneCopy No. in prostate cancer.

FIG. 18. A photograph showing FITC labeled 1G8 antibodies strongly bindPSCA protein on PSCA transfected LNCAP cells.

FIG. 19. A photograph showing FITC labeled 1G8 antibodies weakly bindPreC cells.

FIG. 20. A photograph showing in situ RNA hybridization of PSCA innormal prostate basal cells.

FIG. 21. PSCA immunostaining in primary prostate cancers. Representativeparaffin-embedded sections from four patients were stained withanti-PSCA mAbs. The specimen from patient 1 demonstrates overexpressionof PSCA protein in a Gleason grade 4 tumor (arrow) and undetectableexpression of PSCA in adjacent normal glands (arrowhead) using PSCA mAb1G8. The positively staining cancer completely surrounds the normalglands. The specimen from patient 2 demonstrates heterogeneous stainingin a Gleason grade 3+3/4 cancer. The Gleason pattern 3 glands(arrowhead) stain weakly compared with the larger, more cribriformappearing Gleason pattern 3/4 glands (arrow). The specimen from patient3 demonstrates strong expression of PSCA by a poorly differentiatedGleason 5 (arrow) tumor with mAb 1G8. Patient 4 is a biopsy specimenshowing no PSCA staining in the majority of a poorly differentiatedtumor (arrowhead) and extremely weak staining in a cribriform focusidentified in the specimen. The matched bone metastasis from patient 4is shown in FIG. 28.

FIG. 22. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by biotinylated 1G8 monoclonal antibodylinked to horseradish peroxidase-conjugated streptavidin.

FIG. 23. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by biotinylated 1G8 monoclonal antibodylinked to horseradish peroxidase-conjugated streptavidin.

FIG. 24. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by biotinylated 3E6 monoclonal antibodylinked to horseradish peroxidase-conjugated streptavidin.

FIG. 25. A northern blot showing increased level of PSCA RNA in LAPC9and transitional cell carcinoma of an advanced bladder carcinoma.

FIG. 26. A photograph of a tissue undergoing early stage prostate canceras determined by biotinylated 3E6 monoclonal antibody linked tohorseradish peroxidase-conjugated streptavidin.

FIG. 27. A photograph of a bone sample showing bone metastases ofprostate cancer as determined by hematoxylin stained 3E6 monoclonalantibody.

FIG. 28. PSCA immunostaining in prostate cancer bone metastases. The toppanel shows the hematoxylin and eosin (left) and PSCA (right) stainingof a bony lesion from patient 5. A single focus suspicious for cancer(arrow) was identified in the H and E section and confirmed by intensestaining with anti-PSCA mAb 1G8 (arrow). The bottom panel shows the Hand E (left) and PSCA staining of a bone lesion from patient 4. Theprimary lesion from patient 4 is depicted in FIG. 21. Both the H and Eand PSCA stains show diffuse bony involvement by prostate cancer(arrows). Again, PSCA immunostaining in the bone metastasis is uniformand intense.

FIG. 29. A photograph of a tissue undergoing early stage prostate canceras determined by biotinylated 1G8 monoclonal antibody linked tohorseradish peroxidase-conjugated streptavidin.

FIG. 30. A photograph showing that 1G8 binds LAPC9 cells as determinedby hematoxylin staining.

FIG. 31. A photograph showing that 1G8 binds PSCA-transfected LnCaPcells.

FIG. 32. A photograph showing that 1G8 does not bind LnCaP cells (nottransfected with PSCA).

FIG. 33. Flow cytometric recognition of PSCA on the cell surface ofnonpermeabilized LAPC-9 human prostate cancer cells using mAbs 1G8, 2H9,3E6, 3C5 and 4A10. Staining was compared to an irrelevant isotypecontrol antibody.

FIG. 34. A photograph showing 293T cells transiently transfected withPSCA and immunoblotted with PSCA monoclonal antibodies. Monoclonalantibodies 2H9 and 3E6 binds deglycosylated PSCA but does not bindglycosylated PSCA in 293T cells. In contrast, monoclonal antibodies 1G8,3C5, and 4A10 recognizes glycosylated PSCA.

FIG. 35. Immunofluorescent analysis demonstrating cell surfaceexpression of PSCA in nonpermeabilized prostate cancer cells. LNCaPcells were stably transfected with PSCA and stained with mAbs 1G8, 3E6,3C5 and 4A10. Negative controls included irrelevant isotype antibody andLNCaP cells transfected with control vector, all of which showed nostaining even after prolonged exposures.

FIG. 36. A photograph showing monoclonal antibody 2H9 binds LAPC9 cells.

FIG. 37. A photograph showing immunological reactivity of anti-PSCAmAbs. Immunoprecipitation of PSCA from 293T cells transientlytransfected with PSCA and immunoblot analysis using mAbs 1G8, 2H9, 3C5,3E6 and 4A10. The control was an irrelevant murine IgG mAb. mAbs 1G8,3C5 and 4A10 all recognize equivalent molecular forms of PSCA, whereasmAbs 2H9 and 3E6 only weakly recognize deglycosylated forms of PSCA in293T-PSCA cells in this assay.

FIG. 38. Immunohistochemical staining of normal prostate with anti-PSCAmAbs. Examples shown include a normal gland stained with an irrelevantisotype antibody as a negative control (arrow), PSCA mAb 3E6 and mAb1G8. PSCA mAb 3E6 preferentially stains basal cells (arrow) whencompared with secretory cells (arrowhead), whereas mAb 1G8 stains bothbasal (arrow) and secretory (arrowhead) cells equally. Also shown isstrong staining of an atrophic single-layered gland from a normalprostate specimen stained with PSCA mAb 2H9.

FIG. 39A-B. Expression of PSCA protein in normal tissues. (FIG. 39A)Panel a shows staining of bladder transitional epithelium with mAb 1G8.Panel b shows colonic neuroendocrine cell staining with mAb 1G8. Doublestaining with chromogranin confirmed that the positive cells are ofneuroendocrine origin (not shown). Panel c shows staining of collectingducts (arrow) and tubules with mAb 3E6. Panel d show staining ofplacental trophoblasts with mAb 3E6. (FIG. 39B) Northern blot analysisof PSCA mRNA expression. Total RNA from normal prostate, kidney, bladderand the LAPC-9 prostate cancer xenograft was analyzed using a PSCAspecific probe (top panel). The same membrane was probed with actin tocontrol of loading differences (bottom panel).

FIG. 40A-B. Targeting of mouse PSCA gene. (FIG. 40A) A schematic drawingshowing a strategy for creating a PSCA targeting vector. (FIG. 40B) Aphotograph of a southern blot analysis of genomic DNA using 3′ probeshowing recovery of wild-type (+/+) and heterozygous (+/−) ES cells.

FIG. 41. The upper panel is a schematic drawing of a strategy forgenerating transgenic mouse models of prostate cancer. The lower panelis a list of existing transgenic mouse models of prostate cancer.

FIG. 42. A schematic drawing showing reporter gene constructs fortransfection assay.

FIG. 43. A bar graph showing the tissue-predominant expression (prostateand bladder cells) of the 9 kb human PSCA upstream regulatory regionhaving increased gene expression activity.

FIG. 44. Bar graphs identifying prostate-predominant expression elementswithin PSCA upstream regions having increased gene expression activity,i.e., the 9 kb, 6 kb, 3 kb, and 1 kb PSCA regions.

FIG. 45. A schematic drawing showing the design of transgenic vectorscontaining either a 9 kb or 6 kb human PSCA upstream region operativelylinked to a detectable marker.

FIG. 46. Photographs showing that the 9 kb PSCA upstream region drivesreporter gene expression in prostate, bladder and skin in vivo.

FIG. 47. Photographs of multiple tissue northern blot analysis showingtissue specific expression patterns of human and murine PSCA RNA.

FIG. 48. Complete inhibition of LAPC-9 prostate tumor growth in SCIDmice by treatment with anti-PSCA monoclonal antibodies. The upper panelrepresents mice injected with LAPC-9 s.c. and treated with a mouse IgGcontrol, while in the lower panel mice were injected with LAPC-9 s.c.but treated with the anti-PSCA mAb cocktail. Each data point representsthe ellipsoidal volume of tumors at specified time points as describedin Example 18-A, infra. In the anti-PSCA group, an arbitrary value of 20was given for all data points to create a line, although the actualtumor volume was 0 (Example 18-A, infra).

FIG. 49A-B. Characteristics of anti-PSCA monoclonal antibodies utilizedin the in vivo tumor challenge study described in Example 18. (FIG. 49A)Isotype and epitope map: The region of PSCA protein recognized by theanti-PSCA mAbs was determined by ELISA analysis using GST-fusionproteins (50 ng/well) encoding the indicated amino acids of PSCA.Following incubation of wells with hybridoma supernatants,anti-mouse-HRP conjugate antibody was added and reactivity wasdetermined by the addition of 3,3′ 5,5′-Tetramethylbenzidine base (TMB)substrate. Optical densities (450 nm) are the means of duplicatedeterminations. (FIG. 49B) Epitope map determined by Western analysis:50 ng of the indicated GST-PSCA fusion protein was separated by SDS-PAGEand transferred to nitrocellulose. Western analysis was carried out byincubation of blots with hybridoma supernatants followed byanti-mouse-HRP secondary Ab and visualized by enhanced chemiluminesence.

FIG. 50A-B. Schematic representations of PSCA Capture ELISA. (FIG. 50A)Standardization and control antigens: A GST-fusion protein encodingamino acids 18-98 of the PSCA protein is used for generating a standardcurve for quantification of unknown samples. Also depicted areapproximate epitope binding regions of the anti-PSCA monoclonal andpolyclonal antibodies used in the ELISA. A secreted recombinantmammalian expressed form of PSCA is used for quality control of theELISA assay. This protein contains an Ig leader sequence to directsecretion of the recombinant protein and MYC and 6×HIS (SEQ ID NO:16)epitope tags for affinity purification. (FIG. 50B) ELISA formatschematic.

FIG. 51A-B. Quantification of recombinant secreted PSCA protein. (FIG.51A) PSCA capture ELISA standard curve. (FIG. 51B) Quantification ofPSCA protein secreted by mammalian cells. 2 day conditioned tissueculture supernatants from either 293T cells transfected with emptyvector or with vector encoding recombinant secreted PSCA (secPSCA) wasmixed with an equal volume of either PBS or normal human serum (OmegaScientific) and analyzed for the presence of PSCA protein. Data are themeans of duplicate determinations±range. ND not detectable.

FIG. 52. Immunohistochemical Analysis of cell pellet, LAPC9AD xenograft,a BPH sample, and a prostate carcinoma tissue using anti-PSCA monoclonalantibody 3C5.

FIG. 53. Inhibition of LAPC-9 tumor growth by anti-PSCA monoclonalantibodies. The top panel represents mice injected with 1×10⁶ LAPC-9s.c. and treated with a mouse IgG control (n=10), the middle panelrepresents mice injected with LAPC-9 s.c. and treated with anti-PSCA mAbcocktail (n=10), the bottom panel represents mice injected with LAPC-9s.c. and treated with bovine IgG (n=5). Each data point represents theellipsoidal volume of tumors at specified time points as described inExample 18-B.

FIG. 54. Inhibition of LAPC-9 tumor growth by the anti-PSCA monoclonalantibody 1G8. The upper panel represents mice injected with 1×10⁶ LAPC-9s.c. and treated with a mouse IgG control (n=6), while in the lowerpanel mice were injected with LAPC-9 s.c. but treated with the anti-PSCAmAb 1G8 (n=7). Each data point represents the ellipsoidal volume oftumors at specified time points.

FIG. 55. Inhibition of LAPC-9 tumor growth by anti-PSCA monoclonalantibodies 2A2 and 2H9. The upper panel represents mice injected with1×10⁶ LAPC-9 s.c. and treated with either a mouse IgG control (n=6) orthe 2A2 mAb (n=7). The lower panel represents mice injected with LAPC-9s.c. and treated with the same mouse IgG control (n=6) or the 2H9 mAb(n=7). All data points represent the mean ellipsoidal volume of tumors(mm³) at the specified time points. Error bars represent standard errorof the mean (SEM).

FIG. 56. Circulating PSA levels in LAPC-9 tumor-injected mice aftertreatment with anti-PSCA mAbs 2A2 and 2H9. The upper panel representsthe mice injected with 1×10⁶ LAPC-9 s.c. and treated with either themouse IgG control (n=6) or the 2A2 mAb (n=7). The lower panel representsmice injected with LAPC-9 s.c. but treated with either the same mouseIgG control (n=6) or the 2H9 mAb (n=7). Each data point represents themean PSA level determined from the serum of mice at weekly time points.Error bars represent standard error of the mean (SEM).

FIG. 57. Inhibition of established LAPC-9 prostate cancer xenografts byPSCA monoclonal antibody 3C5. See Example 18-C4 for details.

FIG. 58. Nucleotide sequence (SEQ ID NO:22) and amino acid sequence (SEQID NO:23) of the heavy chain variable domain regions of PSCA monoclonalantibodies 1G8. CDRs are labeled and underlined.

FIG. 59. Nucleotide sequence (SEQ ID NO:24) and amino acid sequence (SEQID NO:25) of the heavy chain variable domain regions of PSCA monoclonalantibodies 4A10. CDRs are labeled and underlined.

FIG. 60. Nucleotide sequence (SEQ ID NO:26) and amino acid sequence (SEQID NO:27) of the heavy chain variable domain regions of PSCA monoclonalantibodies 2H9. CDRs are labeled and underlined.

FIG. 61. Amino acid sequence alignments of CDRs of PSCA mAbs 1G8(CDR1=SEQ ID NO:28; CDR2=SEQ ID NO:31), 4A10 (CDR1=SEQ ID NO:30;CDR2=SEQ ID NO:33; CDR3=SEQ ID NO:35) and 2H9 (CDR1=SEQ ID NO:29;CDR2=SEQ ID NO:32; CDR3=SEQ ID NO:34).

FIG. 62A-D. Photographs showing PSCA protein expression in (FIG. 62A)normal bladder tissue, (FIG. 62B) non-invasive superficial papillarbladder tissue, (FIG. 62C) carcinoma in situ bladder tissue, and (FIG.62D) invasive bladder cancer tissue using immunohistochemical stainingof paraffin-embedded samples with PSCA mAb 1G8.

FIG. 63. Northern blot analysis of PSCA expression in several pancreaticcancer cells lines. Northern blot analysis of PSCA expression in normalprostate and several prostate cancer xenografts are shown alongside forcomparison. RNA levels between all samples were normalized.

FIG. 64. Western blot analysis of PSCA protein expression in prostateand pancreatic cancer cell line using PSCA mab 1G8.

FIG. 65. PSCA mAbs exert growth inhibitory effect through PSCA protein.The growth inhibitory effect of PSCA mAb 1G8 on LAPC-9 and PC-3 prostatetumors is compared, showing no effect on PC-3 tumors, which do notexpress PSCA antigen, but significant growth inhibition in LAPC-9tumors, which do express PSCA antigen. See Examples 18-C1, -C3 fordetails.

FIG. 66A-B. Growth inhibition of established LAPC-9 (AD) orthotopictumors by the anti-PSCA mAb 1G8. (FIG. 66A) Mice having low levels ofserum PSA. Two mg of 1G8 was administered to these mice on days 10, 13,and 15, followed by one mg on days 17, 20, 22, 25, 27, 29, 34, 41, and49 as indicated by the arrows. (FIG. 66B) Mice having moderate levels ofserum PSA. One mg of 1G8 was administered on days 12, 13, 14, 19, 20,22, 25, 27, 29, and 33 as indicated by the arrows.

FIG. 67A-B. Treatment with the anti-PSCA mAb, 1G8, increases survival ofmice bearing established LAPC-9 (AD) orthotopic tumors. (FIG. 67A) Themice in FIG. 66 A, which were treated with 1G8, exhibited an increase insurvival compared to mice treated with PBS. (FIG. 67B) The mice in FIG.66 B, which were treated with 1G8, exhibited an increase in survivalcompared to mice treated with PBS.

FIG. 68A-B. Growth inhibition of established LAPC-9 AD orthotopic tumorsby the anti-PSCA mAb 3C5. (FIG. 68A) One mg of 3C5 was administered totumor-bearing mice on days 6, 8, 10, 13, 15, 17, 20, 22, 24, and 29 asindicated by the arrows. The mice were bled on the days indicated on theX-axis for PSA determinations. (FIG. 68B) Two mg of 3C5 was administeredto tumor-bearing mice on days 9, 12, and 15, followed by one mg on days18, 20, 22, 25, 27, and 29 as indicated by the arrows. The mice werebled on the days indicated on the X-axis for PSA determinations.

FIG. 69A-B. Treatment with the anti-PSCA mAb, 3C5, increases survival ofmice bearing LAPC-9 AD orthotopic tumors. (FIG. 69A) The mice in FIG. 68A, which were treated with 3C5, exhibited an increase in survivalcompared to mice treated with PBS. There were 4 mice in the PBS-treatedgroup and 5 mice in the 3C5-treated group. (FIG. 69B) The mice in FIG.68 B, which were treated with 3C5, exhibited an increase in survivalcompared to mice treated with PBS. There were 6 mice in both thePBS-treated and 3C5-treated groups.

FIG. 70. Growth inhibition of established PC3-PSCA tumors by 1G8 aloneor in combination with doxorubicin. One mg of 1G8 was administered totumor-bearing mice on days 9, 11, 14, 16, 18, 21, 23, 25, and 28 asindicated by the arrows. Twenty-five micrograms of doxorubicin wasadministered on days 9, 16, and 23 as indicated by the (•) symbol.

FIG. 71. Anti-PSCA antibody administered to tumor-bearing micecirculates and targets tumors expressing PSCA Top panel:Immunohistochemistry of a tumor explant from a mouse, bearing anestablished PSCA-expressing tumor, treated with 3C5. Bottom panel:Immunohistochemistry of a tumor explant from a mouse, bearing anestablished PSCA-expressing tumor, treated with mouse IgG.

FIG. 72. Anti-PSCA antibody administered to a tumor-bearing mousecirculates and targets tumors expressing PSCA. A Western blot analysisof tumor lysates from tumors explanted from mice described in FIG. 71,probed with goat anti-mouse IgG-HRP antibody.

FIG. 73. Anti-PSCA antibody administered to a tumor-bearing mousecirculates and targets tumors expressing PSCA. A Western blot analysisof tumor lysates from tumors explanted from mice bearing establishedPSCA-expressing tumors, treated with 1G8. The blot was probed with goatanti-mouse IgG-HRP antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Prostate Stem Cell Antigen (hereinafter“PSCA”). PSCA is a novel, glycosylphosphatidylinositol (GPI)-anchoredcell surface antigen which is expressed in normal cells such prostatecells, urothelium, renal collecting ducts, colonic neuroendocrine cells,placenta, normal bladder and urethral transitional epithelial cells(FIG. 16A-B). PSCA, in addition to normal cells, is also overexpressedby both androgen-dependent and androgen-independent prostate cancercells (FIG. 9-11), prostate cancer metastases to bone (FIGS. 20-24 and26-32), bladder carcinomas (FIGS. 6, 25 and 62), and pancreaticcarcinomas (FIGS. 63 and 64). The expression of PSCA in cancer, e.g.,prostate cancer and bladder cancer, appears to correlate with increasinggrade. Further, overexpression of PSCA (i.e. higher expression thanfound in normal cells) in patients suffering from cancer, e.g., prostatecancer, appears to be indicative of poor prognosis. PSCA mRNA is alsoexpressed by a subset of basal cells in normal prostate. The basal cellepithelium is believed to contain the progenitor cells for theterminally differentiated secretory cells (Bonkhoff et al., 1994,Prostate 24: 114-118). Recent studies using cytokeratin markers suggestthat the basal cell epithelium contains at least two distinct cellularsubpopulations, one expressing cytokeratins 5 and 14 and the othercytokeratins 5, 8 and 18 (Bonkhoff and Remberger, 1996, Prostate 28:98-106). The finding that PSCA is expressed by only a subset of basalcells suggests that PSCA may be a marker for one of these two basal celllineages.

The biological function of PSCA is unknown. The Ly-6 gene family isinvolved in diverse cellular functions, including signal transductionand cell-cell adhesion. Signaling through SCA-2 has been demonstrated toprevent apoptosis in immature thymocytes (Noda et al., 1996, J. Exp.Med. 183: 2355-2360). Thy-1 is involved in T cell activation andtransmits signals through src-like tyrosine kinases (Thomas et al.,1992, J. Biol. Chem. 267: 12317-12322). Ly-6 genes have been implicatedboth in tumorigenesis and in homotypic cell adhesion (Bamezai and Rock,1995, Proc. Natl. Acad. Sci. USA 92: 4294-4298; Katz et al., 1994, Int.J. Cancer 59: 684-691; Brakenhoff et al., 1995, J. Cell Biol. 129:1677-1689). Based on its restricted expression in basal cells and itshomology to Sca-2, we hypothesize that PSCA may play a role instem/progenitor cell functions such as self-renewal (anti-apoptosis)and/or proliferation.

PSCA is highly conserved in mice and humans. The identification of aconserved gene which is predominantly restricted to prostate supportsthe hypothesis that PSCA may play an important role in normal prostatedevelopment.

In its various aspects, as described in detail below, the presentinvention provides PSCA proteins, antibodies, nucleic acid molecules,recombinant DNA molecules, transformed host cells, generation methods,assays, immunotherapeutic methods, transgenic animals, immunological andnucleic acid-based assays, and compositions.

PSCA Proteins

One aspect of the invention provides various PSCA proteins and peptidefragments thereof. As used herein, PSCA refers to a protein that has theamino acid sequence of human PSCA as provided in FIGS. 1B and 3, theamino acid sequence of the murine PSCA homologue as provided in FIG. 3,or the amino acid sequence of other mammalian PSCA homologues, as wellas allelic variants and conservative substitution mutants of theseproteins that have PSCA activity. The PSCA proteins of the inventioninclude the specifically identified and characterized variants hereindescribed, as well as allelic variants, conservative substitutionvariants and homologs that can be isolated/generated and characterizedwithout undue experimentation following the methods outlined below. Forthe sake of convenience, all PSCA proteins will be collectively referredto as the PSCA proteins, the proteins of the invention, or PSCA.

The term “PSCA” includes all naturally occurring allelic variants,isoforms, and precursors of human PSCA as provided in FIGS. 1B and 3 andmurine PSCA as provided in FIG. 3. In general, for example, naturallyoccurring allelic variants of human PSCA will share significant homology(e.g., 70-90%) to the PSCA amino acid sequence provided in FIGS. 1B and3. Allelic variants, though possessing a slightly different amino acidsequence, may be expressed on the surface of prostate cells as a GPIlinked protein or may be secreted or shed. Typically, allelic variantsof the PSCA protein will contain conservative amino acid substitutionsfrom the PSCA sequence herein described or will contain a substitutionof an amino acid from a corresponding position in a PSCA homologue suchas, for example, the murine PSCA homologue described herein.

One class of PSCA allelic variants will be proteins that share a highdegree of homology with at least a small region of the PSCA amino acidsequences presented in FIGS. 1B and 3, but will further contain aradical departure from the sequence, such as a non-conservativesubstitution, truncation, insertion or frame shift. Such alleles aretermed mutant alleles of PSCA and represent proteins that typically donot perform the same biological functions.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

The amino acid sequence of human PSCA protein is provided in FIGS. 1Band 3. Human PSCA is comprised of a single subunit of 123 amino acidsand contains an amino-terminal signal sequence, a carboxy-terminalGPI-anchoring sequence, and multiple N-glycosylation sites. PSCA shows30% homology to stem cell antigen-2 (SCA-2), a member of the Thy-1/Ly-6gene family, a group of cell surface proteins which mark the earliestphases of hematopoetic development. The amino acid sequence of a murinePSCA homologue is shown in FIG. 3. Murine PSCA is a single subunitprotein of 123 amino acids having approximately 70% homology to humanPSCA and similar structural organization.

PSCA proteins may be embodied in many forms, preferably in an isolatedform. As used herein, a protein is said to be isolated when physical,mechanical or chemical methods are employed to remove the PSCA proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated PSCA protein. A purified PSCA proteinmolecule will be substantially free of other proteins or molecules thatimpair the binding of PSCA to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of the PSCA protein include a purified PSCA protein and afunctional, soluble PSCA protein. One example of a functional solublePSCA protein has the amino acid sequence shown in FIG. 1B or a fragmentthereof. In one form, such functional, soluble PSCA proteins orfragments thereof retain the ability to bind antibody or other ligand.

The invention also provides peptides comprising biologically activefragments of the human and murine PSCA amino acid sequences shown inFIGS. 1B and 3. For example, the invention provides a peptide fragmenthaving the amino acid sequence TARIRAVGLLTVISK (SEQ ID NO:9), a peptidefragment having the amino acid sequence VDDSQDYYVGKK (SEQ ID NO:10), andSLNCVDDSQDYYVGK (SEQ ID NO:11).

The peptides of the invention exhibit properties of PSCA, such as theability to elicit the generation of antibodies that specifically bind anepitope associated with PSCA. Such peptide fragments of the PSCAproteins can be generated using standard peptide synthesis technologyand the amino acid sequences of the human or murine PSCA proteinsdisclosed herein. Alternatively, recombinant methods can be used togenerate nucleic acid molecules that encode a fragment of the PSCAprotein. In this regard, the PSCA-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of PSCA.

As discussed below, peptide fragments of PSCA are particularly usefulin: generating domain specific antibodies; identifying agents that bindto PSCA or a PSCA domain; identifying cellular factors that bind to PSCAor a PSCA domain; and isolating homologs or other allelic forms of humanPSCA. PSCA peptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. As examples,hydrophobicity and Chou-Fasman plots of human PSCA are provided in FIGS.4 and 5, respectively. Fragments containing such residues areparticularly useful in generating subunit specific anti-PSCA antibodiesor in identifying cellular factors that bind to PSCA.

Various regions of the PSCA protein can bind to anti-PSCA antibodies.The regions of the PSCA protein may include, for example, the N-terminalregion, middle region, and C-terminal region (Example 18, FIG. 49). TheN-terminal region includes any portion of the PSCA protein encompassedby amino acid residues 2-50, preferably residues 18-50. The middleregion includes any portion of the PSCA protein encompassed by aminoacid residues 46-109, preferably residues 46-98. The C-terminal regionincludes any portion of the PSCA protein encompassed by amino acidresidues 85-123, preferably residues 85-98.

The PSCA proteins of the invention may be useful for a variety ofpurposes, including but not limited to their use as diagnostic and/orprognostic markers of prostate cancer, the ability to elicit thegeneration of antibodies, and as targets for various therapeuticmodalities, as further described below. PSCA proteins may also be usedto identify and isolate ligands and other agents that bind to PSCA. Inthe normal prostate, PSCA is expressed exclusively in a subset of basalcells, suggesting that PSCA may be used as a marker for a specific celllineage within basal epithelium. In addition, the results herein suggestthat this set of basal cells represent the target of neoplastictransformation. Accordingly for example, therapeutic strategies designedto eliminate or modulate the molecular factors responsible fortransformation may be specifically directed to this population of cellsvia the PSCA cell surface protein.

PSCA Antibodies

The invention further provides antibodies (e.g., polyclonal, monoclonal,chimeric, and humanized antibodies) that bind to PSCA. The mostpreferred antibodies will selectively bind to PSCA and will not bind (orwill bind weakly) to non-PSCA proteins. The most preferred antibodieswill specifically bind to PSCA. It is intended that the term“specifically bind” means that the antibody predominantly binds to PSCA.Anti-PSCA antibodies that are particularly contemplated includemonoclonal and polyclonal antibodies as well as fragments thereof (e.g.,recombinant proteins) containing the antigen binding domain and/or oneor more complement determining regions of these antibodies. Theseantibodies can be from any source, e.g., rat, dog, cat, pig, horse,mouse or human.

In one embodiment, the PSCA antibodies specifically bind to theextracellular domain of a PSCA protein, e.g., on the cell surface ofprostate cancer cells from primary lesions and prostate cancer bonemetastases. It is intended that the term “extracellular domain” meansany portion of the PSCA protein which is exterior to the plasma membraneof the cell. In other embodiments, the PSCA antibodies specifically bindto other domains of a PSCA protein or precursor (such as a portion ofthe N-terminal region, the middle region, or the C-terminal region; FIG.49A). As will be understood by those skilled in the art, the regions orepitopes of a PSCA protein to which an antibody is directed may varywith the intended application. For example, antibodies intended for usein an immunoassay for the detection of membrane-bound PSCA on viableprostate cancer cells should be directed to an accessible epitope onmembrane-bound PSCA. Examples of such antibodies are described theExamples which follow. Antibodies that recognize other epitopes may beuseful for the identification of PSCA within damaged or dying cells, forthe detection of secreted PSCA proteins or fragments thereof. Theinvention also encompasses antibody fragments that specificallyrecognize a PSCA protein. As used herein, an antibody fragment isdefined as at least a portion of the variable region of theimmunoglobulin molecule that binds to its target, i.e., the antigenbinding region. Some of the constant region of the immunoglobulin may beincluded.

For example, the overexpression of PSCA in both androgen-dependent andandrogen-independent prostate cancer cells, and the cell surfacelocation of PSCA represent characteristics of an excellent marker forscreening, diagnosis, prognosis, and follow-up assays and imagingmethods. In addition, these characteristics indicate that PSCA may be anexcellent target for therapeutic methods such as targeted antibodytherapy, immunotherapy, and gene therapy.

PSCA antibodies of the invention may be particularly useful indiagnostic assays, imaging methodologies, and therapeutic methods in themanagement of prostate cancer. The invention provides variousimmunological assays useful for the detection of PSCA proteins and forthe diagnosis of prostate cancer. Such assays generally comprise one ormore PSCA antibodies capable of recognizing and binding a PSCA protein,and include various immunological assay formats well known in the art,including but not limited to various types of precipitation,agglutination, complement fixation, radioimmunoassays (MA),enzyme-linked immunosorbent assays (ELISA), enzyme-linkedimmunofluorescent assays (ELIFA) (H. Liu et al. Cancer Research 58:4055-4060 (1998), immunohistochemical analysis and the like. Inaddition, immunological imaging methods capable of detecting prostatecancer are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled PSCA antibodies. Suchassays may be clinically useful in the detection, monitoring, andprognosis of prostate cancer.

In one embodiment, PSCA antibodies and fragments thereof (e.g., Fv,Fab′, F(ab′)2) are used for detecting the presence of a prostate cancer,bladder carcinoma, pancreatic carcinoma, bone metastases of prostatecancer, PIN, or basal epithelial cell expressing a PSCA protein. Thepresence of such PSCA positive (+) cells within various biologicalsamples, including serum, prostate and other tissue biopsy specimens,other tissues such as bone, urine, etc., may be detected with PSCAantibodies. In addition, PSCA antibodies may be used in various imagingmethodologies, such as immunoscintigraphy with Induim-111 (or otherisotope) conjugated antibody. For example, an imaging protocol similarto the one recently described using an In-111 conjugated anti-PSMAantibody may be used to detect recurrent and metastatic prostatecarcinomas (Sodee et al., 1997, Clin Nuc Med 21: 759-766). In relationto other markers of prostate cancer, such as PSMA for example, PSCA maybe particularly useful for targeting androgen independent prostatecancer cells. PSCA antibodies may also be used therapeutically toinhibit PSCA function.

PSCA antibodies may also be used in methods for purifying PSCA proteinsand peptides and for isolating PSCA homologues and related molecules.For example, in one embodiment, the method of purifying a PSCA proteincomprises incubating a PSCA antibody, which has been coupled to a solidmatrix, with a lysate or other solution containing PSCA under conditionswhich permit the PSCA antibody to bind to PSCA; washing the solid matrixto eliminate impurities; and eluting the PSCA from the coupled antibody.Additionally, PSCA antibodies may be used to isolate PSCA positive cellsusing cell sorting and purification techniques. The presence of PSCA onprostate tumor cells (alone or in combination with other cell surfacemarkers) may be used to distinguish and isolate human prostate cancercells from other cells. In particular, PSCA antibodies may be used toisolate prostate cancer cells from xenograft tumor tissue, from cells inculture, etc., using antibody-based cell sorting or affinitypurification techniques. Other uses of the PSCA antibodies of theinvention include generating anti-idiotypic antibodies that mimic thePSCA protein, e.g., a monoclonal anti-idiotypic antibody reactive withan idiotype on any of the monoclonal antibodies of the invention such as1G8, 2A2, 2H9, 3C5, 3E6, 3G3, and 4A10.

The ability to generate large quantities of relatively pure human PSCApositive prostate cancer cells which can be grown in tissue culture oras xenograft tumors in animal models (e.g., SCID or other immunedeficient mice) provides many advantages, including, for example,permitting the evaluation of various transgenes or candidate therapeuticcompounds on the growth or other phenotypic characteristics of arelatively homogeneous population of prostate cancer cells.Additionally, this feature of the invention also permits the isolationof highly enriched preparations of human PSCA positive prostate cancerspecific nucleic acids in quantities sufficient for various molecularmanipulations. For example, large quantities of such nucleic acidpreparations will assist in the identification of rare genes withbiological relevance to prostate cancer disease progression.

Another valuable application of this aspect of the invention is theability to isolate, analyze and experiment with relatively purepreparations of viable PSCA positive prostate tumor cells cloned fromindividual patients with locally advanced or metastatic disease. In thisway, for example, an individual patient's prostate cancer cells may beexpanded from a limited biopsy sample and then tested for the presenceof diagnostic and prognostic genes, proteins, chromosomal aberrations,gene expression profiles, or other relevant genotypic and phenotypiccharacteristics, without the potentially confounding variable ofcontaminating cells. In addition, such cells may be evaluated forneoplastic aggressiveness and metastatic potential in animal models.Similarly, patient-specific prostate cancer vaccines and cellularimmunotherapeutics may be created from such cell preparations.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a PSCA protein, peptide, or fragment, in isolatedor immunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press,NY (1989)). In addition, fusion proteins of PSCA may also be used, suchas a PSCA GST-fusion protein. Cells expressing or overexpressing PSCAmay also be used for immunizations. Similarly, any cell engineered toexpress PSCA may be used. This strategy may result in the production ofmonoclonal antibodies with enhanced capacities for recognizingendogenous PSCA. For example, using standard technologies described inExample 5 and standard hybridoma protocols (Harlow and Lane, 1988,Antibodies: A Laboratory Manual. (Cold Spring Harbor Press)), hybridomasproducing monoclonal antibodies designated 1G8 (ATCC No. HB-12612), 2A2(ATCC No. HB-12613), 2H9 (ATCC No. HB-12614), 3C5 (ATCC No. HB-12616),3E6 (ATCC No. HB-12618), and 3G3 (ATCC No. HB-12615), 4A10 (ATCC No.HB-12617) were generated. These antibody were deposited on Dec. 11, 1998with the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852.

Chimeric antibodies of the invention are immunoglobulin molecules thatcomprise a human and non-human portion. The antigen combining region(variable region) of a chimeric antibody can be derived from a non-humansource (e.g. murine) and the constant region of the chimeric antibodywhich confers biological effector function to the immunoglobulin can bederived from a human source. The chimeric antibody should have theantigen binding specificity of the non-human antibody molecule and theeffector function conferred by the human antibody molecule.

In general, the procedures used to produce chimeric antibodies caninvolve the following steps:

-   -   a) identifying and cloning the correct gene segment encoding the        antigen binding portion of the antibody molecule; this gene        segment (known as the VDJ, variable, diversity and joining        regions for heavy chains or VJ, variable, joining regions for        light chains or simply as the V or variable region) may be in        either the cDNA or genomic form;    -   b) cloning the gene segments encoding the constant region or        desired part thereof;    -   c) ligating the variable region with the constant region so that        the complete chimeric antibody is encoded in a form that can be        transcribed and translated;    -   d) ligating this construct into a vector containing a selectable        marker and gene control regions such as promoters, enhancers and        poly(A) addition signals;    -   e) amplifying this construct in bacteria;    -   f) introducing this DNA into eukaryotic cells (transfection)        most often mammalian lymphocytes;    -   g) selecting for cells expressing the selectable marker;    -   h) screening for cells expressing the desired chimeric antibody;        and    -   k) testing the antibody for appropriate binding specificity and        effector functions.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins [e.g.anti-TNP: Boulianne et al., Nature 312:643 (1984); and anti-tumorantigens: Sahagan et al., J. Immunol. 137:1066 (1986)]. Likewise,several different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes [Neuberger et al., Nature 312:604 (1984)],immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain [Sharon et al., Nature 309:364(1984); Tan et al., J. Immunol. 135:3565-3567 (1985)]. Additionally,procedures for modifying antibody molecules and for producing chimericantibody molecules using homologous recombination to target genemodification have been described (Fell et al., Proc. Natl. Acad. Sci.USA 86:8507-8511 (1989)).

These antibodies are capable of binding to PSCA, e.g., on the cellsurface of prostate cancer cells, thereby confirming the cell surfacelocalization of PSCA. Because these mAbs recognize epitopes on theexterior of the cell surface, they have utility for prostate cancerdiagnosis and therapy. For example, these mAbs were used to locate sitesof metastatic disease (Example 6). Another possibility is that they maybe used (e.g., systemically) to target prostate cancer cellstherapeutically when used alone or conjugated to a radioisotope or othertoxin.

PSCA mAbs stain the cell surface in a punctate manner (see Example 5),suggesting that PSCA may be localized to specific regions of the cellsurface. GPI-anchored proteins are known to cluster indetergent-insoluble glycolipid-enriched microdomains (DIGS) of the cellsurface. These microdomains, which include caveolae andshingolipid-cholesterol rafts, are believed to play critical roles insignal transduction and molecular transport. Thy-1, a homologue of PSCA,has previously been shown to transmit signals to src kinases throughinteraction in lipid-microdomains. Subcellular fractionation experimentsin our laboratory confirm the presence of PSCA in DIGS.

Additionally, some of the antibodies of the invention are internalizingantibodies, i.e., the antibodies are internalized into the cell upon orafter binding. It is intended that the term “internalize” means that theantibody is taken into the cell. Further, some of the antibodies induceinhibition of PSCA positive cancer cell growth.

A characterization of these antibodies, e.g., in prostate cancerspecimens, demonstrates that PSCA protein is overexpressed in prostatecancers relative to normal cells and its expression appears to beupregulated during prostate cancer progression and metastasis. Theseantibodies are useful in studies of PSCA biology and function, as wellas in vivo targeting of PSCA associated cancers, including, withoutlimitation, human prostate cancer, prostate cancer metastases to bone,bladder carcinomas, and pancreatic carcinomas.

PSCA mAbs which specifically recognize and bind to the extracellulardomain of the PSCA protein are described herein. Some of these have beenshown to bind to native PSCA as expressed on the cell surface and somehave been shown to inhibit the in vivo growth of prostate tumor cells.

The amino acid sequence of PSCA presented herein may be used to selectspecific regions of the PSCA protein for generating antibodies. Forexample, hydrophobicity and hydrophilicity analyses of the PSCA aminoacid sequence may be used to identify hydrophilic regions in the PSCAstructure. Regions of the PSCA protein that show immunogenic structure,as well as other regions and domains, can readily be identified usingvarious other methods known in the art, such as Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis. Fragments containing these residues areparticularly suited in generating specific classes of anti-PSCAantibodies. Particularly useful fragments include, but are not limitedto, the sequences TARIRAVGLLTVISK (SEQ ID NO:9) and SLNCVDDSQDYYVGK (SEQID NO:11).

As described in Example 2, below, a rabbit polyclonal antibody wasgenerated against the former fragment, prepared as a synthetic peptide,and affinity purified using a PSCA-glutathione S transferase fusionprotein. Recognition of PSCA by this antibody was established byimmunoblot and immunoprecipitation analysis of extracts of 293T cellstransfected with PSCA and a GST-PSCA fusion protein. This antibody alsoidentified the cell surface expression of PSCA in PSCA-transfected 293Tand LAPC-4 cells using fluorescence activated cell sorting (FACS).

Additionally, a sheep polyclonal antibody was generated against thelatter fragment, prepared as a synthetic peptide, and affinity purifiedusing a peptide Affi-gel column (also by the method of Example 2).Recognition of PSCA by this antibody was established by immunoblot andimmunoprecipitation analysis of extracts of LNCaP cells transfected withPSCA. This antibody also identified the cell surface expression of PSCAin PSCA-transfected LNCaP cells using fluorescence activated cellsorting (FACS) and immunohistochemistry analysis.

Methods for preparing a protein for use as an immunogen and forpreparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art. In somecircumstances, direct conjugation using, for example, carbodiimidereagents may be used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., may be effective.Administration of a PSCA immunogen is conducted generally by injectionover a suitable time period and with use of a suitable adjuvant, as isgenerally understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, monoclonalantibody preparations are preferred. Immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the PSCA protein or PSCAfragment. When the appropriate immortalized cell culture secreting thedesired antibody is identified, the cells can be cultured either invitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of the monoclonalantibodies of the invention or the polyclonal antisera (e.g., Fab,F(ab′)₂, Fv fragments, fusion proteins) which contain theimmunologically significant portion (i.e., a portion that recognizes andbinds PSCA) can be used as antagonists, as well as the intactantibodies. Humanized antibodies directed against PSCA is also useful.As used herein, a humanized PSCA antibody is an immunoglobulin moleculewhich is capable of binding to PSCA and which comprises a FR regionhaving substantially the amino acid sequence of a human immunoglobulinand a CDR having substantially the amino acid sequence of non-humanimmunoglobulin or a sequence engineered to bind PSCA. Methods forhumanizing murine and other non-human antibodies by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmnan et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Use of immunologically reactive fragments, such as the Fab, Fab′, orF(ab)₂ fragments is often preferable, especially in a therapeuticcontext, as these fragments are generally less immunogenic than thewhole immunoglobulin. Further, bi-specific antibodies specific for twoor more epitopes may be generated using methods generally known in theart. Further, antibody effector functions may be modified so as toenhance the therapeutic effect of PSCA antibodies on cancers. Forexample, cysteine residues may be engineered into the Fc region,permitting the formation of interchain disulfide bonds and thegeneration of homodimers which may have enhanced capacities forinternalization, ADCC and/or complement-mediated cell killing (see, forexample, Caron et al., 1992, J. Exp. Med. 176: 1191-1195; Shopes, 1992,J. Immunol. 148: 2918-2922). Homodimeric antibodies may also begenerated by cross-linking techniques known in the art (e.g., Wolff etal., Cancer Res. 53: 2560-2565). The invention also providespharmaceutical compositions having the monoclonal antibodies oranti-idiotypic monoclonal antibodies of the invention.

The generation of monoclonal antibodies (mAbs) capable of binding tocell surface PSCA are described in Example 5. Epitope mapping of thesemAbs indicates that they recognize different epitopes on the PSCAprotein. For example, one recognizes an epitope within thecarboxy-terminal region and the other recognizing an epitope within theamino-terminal region. Such PSCA antibodies may be particularly wellsuited to use in a sandwich-formatted ELISA, given their differingepitope binding characteristics.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the PSCA protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin. Theinvention includes an antibody, e.g., a monoclonal antibody whichcompetitively inhibits the immunospecific binding of any of themonoclonal antibodies of the invention to PSCA.

Alternatively, methods for producing fully human monoclonal antibodies,include phage display and transgenic methods, are known and may be usedfor the generation of human mAbs (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). For example, fully human anti-PSCAmonoclonal antibodies may be generated using cloning technologiesemploying large human Ig gene combinatorial libraries (i.e., phagedisplay)(Griffiths and Hoogenboom, Building an in vitro immune system:human antibodies from phage display libraries. In: Protein Engineeringof Antibody Molecules for Prophylactic and Therapeutic Applications inMan. Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton andBarbas, Human Antibodies from combinatorial libraries. Id., pp 65-82).Fully human anti-PSCA monoclonal antibodies may also be produced usingtransgenic mice engineered to contain human immunoglobulin gene loci asdescribed in PCT Patent Application WO98/24893, Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of anti-PSCA mAbs against the target antigen may beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,PSCA proteins, peptides, PSCA-expressing cells or extracts thereof.Anti-PSCA mAbs may also be characterized in various in vitro assays,including complement-mediated tumor cell lysis, antibody-dependent cellcytotoxicity (ADCC), antibody-dependent macrophage-mediated cytotoxicity(ADMMC), tumor cell proliferation, etc. Examples of such in vitro assaysare presented in Example 19, infra.

The antibody or fragment thereof of the invention may be cytostatic tothe cell, to which it binds. It is intended that the term “cytostatic”means that the antibody can inhibit growth, but not necessarily kill,PSCA-positive cells.

The antibody or fragment thereof of the invention may be labeled with adetectable marker or conjugated to a second molecule, such as atherapeutic agent (e.g., a cytotoxic agent) thereby resulting in animmunoconjugate. For example, the therapeutic agent includes, but is notlimited to, an anti-tumor drug, a toxin, a radioactive agent, acytokine, a second antibody or an enzyme. Further, the inventionprovides an embodiment wherein the antibody of the invention is linkedto an enzyme that converts a prodrug into a cytotoxic drug.

The immunoconjugate can be used for targeting the second molecule to aPSCA positive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy, inDeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice ofOncology, 4th ed., J.B. Lippincott Co., Philadelphia, 2624-2636).

Examples of cytotoxic agents include, but are not limited to ricin,ricin A-chain, doxorubicin, daunorubicin, taxol, ethiduim bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin,curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor,maytansinoids, and glucocorticoid and other chemotherapeutic agents, aswell as radioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme. Antibodies mayalso be conjugated to an anti-cancer pro-drug activating enzyme capableof converting the pro-drug to its active form. See, for example, U.S.Pat. No. 4,975,287.

Additionally, the recombinant protein of the invention comprising theantigen-binding region of any of the monoclonal antibodies of theinvention can be used to treat cancer. In such a situation, theantigen-binding region of the recombinant protein is joined to at leasta functionally active portion of a second protein having therapeuticactivity. The second protein can include, but is not limited to, anenzyme, lymphokine, oncostatin or toxin. Suitable toxins include thosedescribed above.

Techniques for conjugating or joining therapeutic agents to antibodiesare well known (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)). The use of PSCA antibodies as therapeutic agents is furtherdescribed in the subsection “PROSTATE CANCER IMMUNOTHERAPY” below.

PSCA-Encoding Nucleic Acid Molecules

Another aspect of the invention provides various nucleic acid moleculesencoding PSCA proteins and fragments thereof, preferably in isolatedform, including DNA, RNA, DNA/RNA hybrid, and related molecules, nucleicacid molecules complementary to the PSCA coding sequence or a partthereof, and those which hybridize to the PSCA gene or to PSCA-encodingnucleic acids. Particularly preferred nucleic acid molecules will have anucleotide sequence substantially identical to or complementary to thehuman or murine DNA sequences herein disclosed. Specificallycontemplated are genomic DNA, cDNAs, ribozymes, and antisense molecules,as well as nucleic acids based on an alternative backbone or includingalternative bases, whether derived from natural sources or synthesized.

For example, antisense molecules can be RNAs or other molecules,including peptide nucleic acids (PNAs) or non-nucleic acid moleculessuch as phosphorothioate derivatives, that specifically bind DNA or RNAin a base pair-dependent manner. A skilled artisan can readily obtainthese classes of nucleic acid molecules using the herein described PSCAsequences. For convenience, PSCA-encoding nucleic acid molecules will bereferred to herein as PSCA-encoding nucleic acid molecules, PSCA genes,or PSCA sequences.

The nucleotide sequence of a cDNA encoding one allelic form of humanPSCA is provided in FIG. 1A. The nucleotide sequence of a cDNA encodinga murine PSCA homologue (“murine PSCA”) is provided in FIG. 2. Genomicclones of human and murine PSCA have also been isolated, as described inExample 4. Both the human and murine genomic clones contain three exonsencoding the translated and 3′ untranslated regions of the PSCA gene. Afourth exon encoding a 5′ untranslated region is presumed to exist basedon PSCA's homology to other members of the Ly-6 and Thy-1 gene families(FIG. 8A).

The human PSCA gene maps to chromosome 8q24.2. Human stem cell antigen 2(RIG-E), as well as one other recently identified human Ly-6 homologue(E48) are also localized to this region, suggesting that a large familyof related genes may exist at this locus (Brakenhoff et al., 1995,supra; Mao et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5910-5914).Intriguingly, chromosome 8q has been reported to be a region of allelicgain and amplification in a majority of advanced and recurrent prostatecancers (Cher et al., 1994, Genes Chrom. Cancer 11: 153-162). c-myclocalizes proximal to PSCA at chromosome 8q24 and extra copies of c-myc(either through allelic gain or amplification) have been found in 68% ofprimary prostate tumors and 96% of metastases (Jenkins et al., 1997,Cancer Res. 57: 524-531).

Embodiments of the PSCA-encoding nucleic acid molecules of the inventioninclude primers, which allow the specific amplification of nucleic acidmolecules of the invention or of any specific parts thereof, and probesthat selectively or specifically hybridize to nucleic acid molecules ofthe invention or to any part thereof. The nucleic acid probes can belabeled with a detectable marker. Examples of a detectable markerinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, a chemiluminescent compound, a metal chelatoror an enzyme. Such labeled probes can be used to diagnosis the presenceof a PSCA protein as a means for diagnosing cell expressing a PSCAprotein. Technologies for generating DNA and RNA probes are well known.

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid molecules that encode polypeptides other than PSCA. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated PSCA-encoding nucleic acid molecule.

The invention further provides fragments of the PSCA-encoding nucleicacid molecules of the present invention. As used herein, a fragment of aPSCA-encoding nucleic acid molecule refers to a small portion of theentire PSCA-encoding sequence. The size of the fragment will bedetermined by its intended use.

For example, if the fragment is chosen so as to encode an active portionof the PSCA protein, such an active domain, effector binding site or GPIbinding domain, then the fragment will need to be large enough to encodethe functional region(s) of the PSCA protein. If the fragment is to beused as a nucleic acid probe or PCR primer, then the fragment length ischosen so as to obtain a relatively small number of false positivesduring probing/priming.

Fragments of human PSCA that are particularly useful as selectivehybridization probes or PCR primers can be readily identified from theentire PSCA sequence using art-known methods. One set of PCR primersthat are useful for RT-PCR analysis comprise 5′ primer-TGCTTGCCCTGTTGATGGCAG- (SEQ ID NO:12) and 3′ primer-CCAGAGCAGCAGGCCGAGTGCA- (SEQ ID NO:13).

Methods for Isolating Other PSCA-Encoding Nucleic Acid Molecules

The PSCA-encoding nucleic acid molecules described herein enable theisolation of PSCA homologues, alternatively sliced isoforms, allelicvariants, and mutant forms of the PSCA protein as well as their codingand gene sequences. The most preferred source of PSCA homologs aremammalian organisms.

For example, a portion of the PSCA-encoding sequence herein describedcan be synthesized and used as a probe to retrieve DNA encoding a memberof the PSCA family of proteins from organisms other than human, allelicvariants of the human PSCA protein herein described, and genomicsequence containing the PSCA gene. Oligomers containing approximately18-20 nucleotides (encoding about a 6-7 amino acid stretch) are preparedand used to screen genomic DNA or cDNA libraries to obtain hybridizationunder stringent conditions or conditions of sufficient stringency toeliminate an undue level of false positives. In a particular embodiment,cDNA encoding human PSCA was used to isolate a full length cDNA encodingthe murine PSCA homologue as described in Example 3 herein. The murineclone encodes a protein with 70% amino acid identity to human PSCA.

In addition, the amino acid sequence of the human PSCA protein may beused to generate antibody probes to screen expression libraries preparedfrom cells. Typically, polyclonal antiserum from mammals such as rabbitsimmunized with the purified protein (as described below) or monoclonalantibodies can be used to probe an expression library, prepared from atarget organism, to obtain the appropriate coding sequence for a PSCAhomologue. The cloned cDNA sequence can be expressed as a fusionprotein, expressed directly using its own control sequences, orexpressed by constructing an expression cassette using control sequencesappropriate to the particular host used for expression of the enzyme.

Genomic clones containing PSCA genes may be obtained using molecularcloning methods well known in the art. In one embodiment, a humangenomic clone of approximately 14 kb containing exons 1-4 of the PSCAgene was obtained by screening a lambda phage library with a human PSCAcDNA probe, as more completely described in Example 4 herein. In anotherembodiment, a genomic clone of approximately 10 kb containing the murinePSCA gene was obtained by screening a murine BAC (bacterial artificialchromosome) library with a murine PSCA cDNA (also described in Example4).

Additionally, pairs of oligonucleotide primers can be prepared for usein a polymerase chain reaction (PCR) to selectively amplify/clone aPSCA-encoding nucleic acid molecule, or fragment thereof. A PCRdenature/anneal/extend cycle for using such PCR primers is well known inthe art and can readily be adapted for use in isolating otherPSCA-encoding nucleic acid molecules. Regions of the human PSCA genethat are particularly well suited for use as a probe or as primers canbe readily identified.

Non-human homologues of PSCA, naturally occurring allelic variants ofPSCA and genomic PSCA sequences will share a high degree of homology tothe human PSCA sequences herein described. In general, such nucleic acidmolecules will hybridize to the human PSCA sequence under stringentconditions. Such sequences will typically contain at least 70% homology,preferably at least 80%, most preferably at least 90% homology to thehuman PSCA sequence.

Stringent conditions are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015M NaCl/0.0015M sodiumnitrate/0.1% SDS at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.

Another example is use of 50% formamide, 5×SSC (0.75M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS. A skilled artisan can readily determine andvary the stringency conditions appropriately to obtain a clear anddetectable hybridization signal.

Recombinant DNA Molecules Containing PSCA-Encoding Nucleic Acids

Also provided are recombinant DNA molecules (rDNAs) that contain aPSCA-encoding sequences as herein described, or a fragment thereof. Asused herein, a rDNA molecule is a DNA molecule that has been subjectedto molecular manipulation in vitro. Methods for generating rDNAmolecules are well known in the art, for example, see Sambrook et al.,Molecular Cloning (1989). In the preferred rDNA molecules of the presentinvention, a PSCA-encoding DNA sequence that encodes a PSCA protein or afragment of PSCA, is operably linked to one or more expression controlsequences and/or vector sequences. The rDNA molecule can encode eitherthe entire PSCA protein, or can encode a fragment of the PSCA protein.

The choice of vector and/or expression control sequences to which thePSCA-encoding sequence is operably linked depends directly, as is wellknown in the art, on the functional properties desired, e.g., proteinexpression, and the host cell to be transformed. A vector contemplatedby the present invention is at least capable of directing thereplication or insertion into the host chromosome, and preferably alsoexpression, of the PSCA-encoding sequence included in the rDNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, enhancers, transcription terminators andother regulatory elements. Preferably, an inducible promoter that isreadily controlled, such as being responsive to a nutrient in the hostcell's medium, is used.

In one embodiment, the vector containing a PSCA-encoding nucleic acidmolecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule intrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art. In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the PSCA-encoding sequence in abacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Various viral vectors well known to thoseskilled in the art may also be used, such as, for example, a number ofwell known retroviral vectors.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to variant rDNAmolecules that contain a PSCA-encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. Southern et al., J Mol Anal Genet (1982) 1:327-341.Alternatively, the selectable marker can be present on a separateplasmid, and the two vectors are introduced by cotransfection of thehost cell, and selected by culturing in the presence of the appropriatedrug for the selectable marker.

In accordance with the practice of the invention, the vector can be aplasmid, cosmid or phage vector encoding the cDNA molecule discussedabove. Additionally, the invention provides a host-vector systemcomprising the plasmid, cosmid or phage vector transfected into asuitable eukaryotic host cell. Examples of suitable eukaryotic hostcells include a yeast cell, a plant cell, or an animal cell, such as amammalian cell. Examples of suitable cells include the LnCaP, LAPC-4,and other prostate cancer cell lines. The host-vector system is usefulfor the production of a PSCA protein. Alternatively, the host cell canbe prokaryotic, such as a bacterial cell.

Transformed Host Cells

The invention further provides host cells transformed with a nucleicacid molecule that encodes a PSCA protein or a fragment thereof. Thehost cell can be either prokaryotic or eukaryotic. Eukaryotic cellsuseful for expression of a PSCA protein are not limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of a PSCA gene.Preferred eukaryotic host cells include, but are not limited to, yeast,insect and mammalian cells, preferably vertebrate cells such as thosefrom a mouse, rat, monkey or human fibroblastic cell line. Prostatecancer cell lines, such as the LnCaP and LAPC-4 cell lines may also beused. Any prokaryotic host can be used to express a PSCA-encoding rDNAmolecule. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with an rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see, for example, Cohen etal., Proc Acad Sci USA (1972) 69:2110; and Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982). With regard to transformation of vertebrate cellswith vectors containing rDNAs, electroporation, cationic lipid or salttreatment methods are typically employed, see, for example, Graham etal., Virol (1973) 52:456; Wigler et al., Proc Natl Acad Sci USA (1979)76:1373-76.

Successfully transformed cells, i.e., cells that contain an rDNAmolecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce single colonies.Cells from those colonies can be harvested, lysed and their DNA contentexamined for the presence of the rDNA using a method such as thatdescribed by Southern, J Mol Biol (1975) 98:503, or Berent et al.,Biotech (1985) 3:208 or the proteins produced from the cell assayed viaan immunological method.

Recombinant Methods of Generating PSCA Proteins

The invention further provides methods for producing a PSCA proteinusing one of the PSCA-encoding nucleic acid molecules herein described.In general terms, the production of a recombinant PSCA protein typicallycan involve the following steps (Maniatis, supra).

First, a nucleic acid molecule is obtained that encodes a PSCA proteinor a fragment thereof, such as the nucleic acid molecule depicted inFIG. 1A. The PSCA-encoding nucleic acid molecule is then preferablyplaced in an operable linkage with suitable control sequences, asdescribed above, to generate an expression unit containing thePSCA-encoding sequence. The expression unit is used to transform asuitable host and the transformed host is cultured under conditions thatallow the production of the PSCA protein. Optionally the PSCA protein isisolated from the medium or from the cells; recovery and purification ofthe protein may not be necessary in some instances where some impuritiesmay be tolerated.

Each of the foregoing steps may be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in an appropriate host. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using an appropriate combination of replicons and controlsequences. The control sequences, expression vectors, and transformationmethods are dependent on the type of host cell used to express the geneand were discussed in detail earlier. Suitable restriction sites can, ifnot normally available, be added to the ends of the coding sequence soas to provide an excisable gene to insert into these vectors. A skilledartisan can readily adapt any host/expression system known in the artfor use with PSCA-encoding sequences to produce a PSCA protein.

In a specific embodiment described in the examples which follow, asecreted form of PSCA may be conveniently expressed in 293T cellstransfected with a CMV-driven expression vector encoding PSCA with aC-terminal 6×His (SEQ ID NO:16) and MYC tag (pcDNA3.1/mycHIS,Invitrogen). The secreted HIS-tagged PSCA in the culture media may bepurified using a nickel column using standard techniques.

Assays for Identifying PSCA Ligands and Other Binding Agents

Another aspect of the invention relates to assays and methods that canbe used to detect and identify PSCA ligands and other agents andcellular constituents that bind to PSCA. Specifically, PSCA ligands andother agents and cellular constituents that bind to PSCA can beidentified by the ability of the PSCA ligand or other agent orconstituent to bind to PSCA and/or the ability to inhibit/stimulate PSCAactivity. Assays for PSCA activity (e.g., binding) using a PSCA proteinare suitable for use in high through-put screening methods.

In one embodiment, the assay comprises mixing PSCA with a test agent orcellular extract. After mixing under conditions that allow associationof PSCA with the agent or component of the extract, the mixture isanalyzed to determine if the agent/component is bound to PSCA. Bindingagents/components are identified as being able to bind to PSCA.Alternatively or consecutively, PSCA activity can be directly assessedas a means for identifying agonists and antagonists of PSCA activity.

Alternatively, targets that bind to a PSCA protein can be identifiedusing a yeast two-hybrid system (Fields, S. and Song, O. (1989), Nature340:245-246) or using a binding-capture assay (Harlow, supra). In theyeast two hybrid system, an expression unit encoding a fusion proteinmade up of one subunit of a two subunit transcription factor and thePSCA protein is introduced and expressed in a yeast cell. The cell isfurther modified to contain (1) an expression unit encoding a detectablemarker whose expression requires the two subunit transcription factorfor expression and (2) an expression unit that encodes a fusion proteinmade up of the second subunit of the transcription factor and a clonedsegment of DNA. If the cloned segment of DNA encodes a protein thatbinds to the PSCA protein, the expression results in the interaction ofthe PSCA and the encoded protein. This brings the two subunits of thetranscription factor into binding proximity, allowing reconstitution ofthe transcription factor. This results in the expression of thedetectable marker. The yeast two hybrid system is particularly useful inscreening a library of cDNA encoding segments for cellular bindingpartners of PSCA.

PSCA proteins which may be used in the above assays include, but are notlimited to, an isolated PSCA protein, a fragment of a PSCA protein, acell that has been altered to express a PSCA protein, or a fraction of acell that has been altered to express a PSCA protein. Further, the PSCAprotein can be the entire PSCA protein or a defined fragment of the PSCAprotein. It will be apparent to one of ordinary skill in the art that solong as the PSCA protein can be assayed for agent binding, e.g., by ashift in molecular weight or activity, the present assay can be used.

The method used to identify whether an agent/cellular component binds toa PSCA protein will be based primarily on the nature of the PSCA proteinused. For example, a gel retardation assay can be used to determinewhether an agent binds to PSCA or a fragment thereof. Alternatively,immunodetection and biochip technologies can be adopted for use with thePSCA protein. A skilled artisan can readily employ numerous art-knowntechniques for determining whether a particular agent binds to a PSCAprotein.

Agents and cellular components can be further tested for the ability tomodulate the activity of a PSCA protein using a cell-free assay systemor a cellular assay system. As the activities of the PSCA protein becomemore defined, functional assays based on the identified activity can beemployed.

As used herein, an agent is said to antagonize PSCA activity when theagent reduces PSCA activity. The preferred antagonist will selectivelyantagonize PSCA, not affecting any other cellular proteins. Further, thepreferred antagonist will reduce PSCA activity by more than 50%, morepreferably by more than 90%, most preferably eliminating all PSCAactivity.

As used herein, an agent is said to agonize PSCA activity when the agentincreases PSCA activity. The preferred agonist will selectively agonizePSCA, not affecting any other cellular proteins. Further, the preferredantagonist will increase PSCA activity by more than 50%, more preferablyby more than 90%, most preferably more than doubling PSCA activity.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences of the PSCA protein. An example of randomlyselected agents is the use of a chemical library or a peptidecombinatorial library, or a growth broth of an organism or plantextract.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis that takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences that make up the PSCAprotein. For example, a rationally selected peptide agent can be apeptide whose amino acid sequence is identical to a fragment of a PSCAprotein.

The agents tested in the methods of the present invention can be, asexamples, peptides, small molecules, and vitamin derivatives, as well ascarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents used in the presentscreening method. One class of agents of the present invention arepeptide agents whose amino acid sequences are chosen based on the aminoacid sequence of the PSCA protein. Small peptide agents can serve ascompetitive inhibitors of PSCA protein assembly.

Peptide agents can be prepared using standard solid phase (or solutionphase) peptide synthesis methods, as is known in the art. In addition,the DNA encoding these peptides may be synthesized using commerciallyavailable oligonucleotide synthesis instrumentation and producedrecombinantly using standard recombinant production systems. Theproduction using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the PSCA protein. As describedabove, antibodies are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the PSCA protein intended to be targeted by the antibodies. Criticalregions may include the domains identified in FIG. 15A-B. Such agentscan be used in competitive binding studies to identify second generationinhibitory agents.

The cellular extracts tested in the methods of the present invention canbe, as examples, aqueous extracts of cells or tissues, organic extractsof cells or tissues or partially purified cellular fractions. A skilledartisan can readily recognize that there is no limit as to the source ofthe cellular extract used in the screening method of the presentinvention.

Agents that bind a PSCA protein, such as a PSCA antibody, can be used tomodulate the activity of PSCA, to target anticancer agents toappropriate mammalian cells, or to identify agents that block theinteraction with PSCA. Cells expressing PSCA can be targeted oridentified by using an agent that binds to PSCA.

How the PSCA binding agents will be used depends on the nature of thePSCA binding agent. For example, a PSCA binding agent can be used to:deliver conjugated toxins, such a diphtheria toxin, cholera toxin, ricinor pseudomonas exotoxin, to a PSCA expressing cell; modulate PSCAactivity; to directly kill PSCA expressing cells; or in screens toidentify competitive binding agents. For example, a PSCA inhibitoryagent can be used to directly inhibit the growth of PSCA expressingcells whereas a PSCA binding agent can be used as a diagnostic agent.

There are multiple diagnostic uses of the invention. For example, theinvention provides methods for diagnosing in a subject, e.g., an animalor human subject, a cancer associated with the presence of the PSCAprotein. In one embodiment, the method comprises quantitativelydetermining the number of PSCA protein in the sample (e.g., cell orbiological fluid sample) using any one or combination of the antibodiesof the invention. Then the number so determined can be compared with theamount in a sample from a normal subject. The presence of a measurablydifferent amount (i.e., the number of PSCA in the test sample exceedsthe number from a normal sample) in the samples indicating the presenceof the cancer. PSCA is overexpressed on a cell when the number of PSCAin the test sample exceeds the number from a normal sample.

In another embodiment, diagnosis involves quantitatively determining ina sample from the subject the amount of RNA encoding the PSCA proteinusing the nucleic acid of the invention. The amount so determined can becompared with the amount of RNA in a sample from a normal subject. Onceagain, the presence of a measurable different amount indicating thepresence of the cancer.

Additionally, the invention provides methods for monitoring the courseof cancer (e.g., prostate, bone metastases of prostate cancer, bladder,pancreatic cancer) or disorders associated with PSCA in a subject bymeasuring the amount of PSCA in a sample from the subject at variouspoints in time. This is done for purposes of determining a change in theamount of PSCA in the sample e.g., to determine whether the change is asmall change in the amount or a large change, i.e., overexpression ofPSCA. In one embodiment, the method comprises quantitatively determiningin a first sample from the subject the presence of a PSCA protein andcomparing the amount so determined with the amount present in a secondsample from the subject, such samples being taken at different points intime, a difference in the amounts determined being indicative of thecourse of the cancer.

In another embodiment, monitoring is effected by quantitativelydetermining in a first sample from the subject the presence of a PSCARNA and comparing the amount so determined with the amount present in asecond sample from the subject, such samples being taken at differentpoints in time, a difference in the amounts determined being indicativeof the course of the cancer (e.g, prostate, bone metastases of prostatecancer, bladder and pancreatic cancer).

As a further embodiment, the diseases or disorders associated with PSCAcan be monitored in a sample by detecting an increase in or increasedPSCA gene copy number. An increase in or increased PSCA gene copy numberis important because it may correlate with poor outcome.

The sample can be from an animal or a human. Further, the sample can bea cell sample. For example, using the methods of the invention, organtissues such as prostate tissue, bladder tissue, pancreatic tissue,neuroendocrine tissue, and bone (any tissue where carcinomas canmetastasize, e.g., node, lung, liver, pancreas) can be evaluated for thepresence of cancer or metastatic lesion. Alternatively, the sample canbe a biological fluid, e.g., urine, blood sera or plasma.

In accordance with the practice of the invention, detection can beeffected by immunologic detection means involving histology, blotting,ELISA, and ELIFA. When the sample is a tissue or cell sample it can beformalin-fixed, paraffin-embedded or frozen.

The invention additionally provides methods of determining a differencein the amount and distribution of PSCA in tissue sections from aneoplastic tissue to be tested relative to the amount and distributionof PSCA in tissue sections from a normal tissue. In one embodiment, themethod comprises contacting both the tissue to be tested and the normaltissue with a monoclonal antibody that specifically forms a complex withPSCA and thereby detecting the difference in the amount and distributionof PSCA.

Further, the invention provides a method for diagnosing a neoplastic orpreneoplastic condition in a subject. This method comprises obtainingfrom the subject a sample of a tissue, detecting a difference in theamount and distribution of PSCA in the using the method above, adistinct measurable difference being indicative of such neoplastic orpreneoplastic condition.

In accordance with the practice of the invention, the antibody can bedirected to the epitope to which any of the monoclonal antibodies of theinvention is directed. Further, the tissue section can be from thebladder, prostate, bone, lymphatic tissues, pancreas, other organs, ormuscle.

The invention also provides methods of detecting and quantitativelydetermining the concentration of PSCA in a biological fluid sample. Inone embodiment the method comprises contacting a solid support with anexcess of one or more monoclonal antibodies which forms (preferablyspecifically forms) a complex with PSCA under conditions permitting themonoclonal antibody to attach to the surface of the solid support. Theresulting solid support to which the monoclonal antibody is attached isthen contacted with a biological fluid sample so that the PSCA in thebiological fluid binds to the antibody and forms a PSCA-antibodycomplex. The complexed can be labeled directly or indirectly with adetectable marker. Alternatively, either the PSCA or the antibody can belabeled before the formation the complex. The complex can then bedetected and quantitatively determined thereby detecting andquantitatively determining the concentration of PSCA in the biologicalfluid sample. A high concentration of PSCA in the sample relative tonormal cells being indicative of a neoplastic or preneoplasticcondition.

In accordance with the practice of the invention, the biological fluidincludes but is not limited to tissue extract, urine, blood, serum, andphlegm. Further, the detectable marker includes but is not limited to anenzyme, biotin, a fluorophore, a chromophore, a heavy metal, aparamagnetic isotope, or a radioisotope.

Further, the invention provides a diagnostic kit comprising an antibodythat recognizes and binds PSCA (an anti-PSCA antibody); and a conjugateof a detectable label and a specific binding partner of the anti-PSCAantibody. In accordance with the practice of the invention the labelincludes, but is not limited to, enzymes, radiolabels, chromophores andfluorescers.

Cancer Immunotherapy

Since PSCA protein is expressed or overexpressed in many cancers,including but not limited to prostate tumors, metastases of prostatetumors (such as bone metastases), bladder cancer and pancreatic cancer,it is a target for cancer immunotherapy. These immunotherapeutic methodsinclude the use of antibody therapy, in vivo vaccines, and ex vivoimmunotherapy approaches.

In one approach, the invention provides PSCA antibodies that may be usedsystemically to treat cancer, such as prostate, bladder and pancreaticcancer. PSCA antibodies may also be useful in the treatment of variousother benign and malignant tumors. Antibodies which bind specifically tothe extracellular domain of PSCA are preferred. Antibodies which targetthe tumor cells but not the surrounding non-tumor cells and tissue arepreferred. Thus, the invention provides a method of treating a patientsusceptible to or having a cancer which expresses PSCA antigen,comprising administering to said patient an effective amount of anantibody which binds specifically to the extracellular domain of PSCA.In another approach, the invention provides a method of inhibiting thegrowth of tumor cells expressing PSCA, comprising administering to apatient an antibody which binds specifically to the extracellular domainof PSCA in an amount effective to inhibit growth of the tumor cells.PSCA mAbs may also be used in a method for selectively inhibiting thegrowth of or killing a cell expressing PSCA antigen comprising reactinga PSCA antibody immunoconjugate or immunotoxin with the cell in anamount sufficient to inhibit the growth of or kill the cell.

For example, unconjugated PSCA antibody (including monoclonal,polyclonal, chimeric, humanized, fully human and fragments thereof(e.g., recombinant proteins)) may be introduced into a patient such thatthe antibody binds to PSCA on cancer cells and mediates growthinhibition of such cells (including the destruction thereof), and thetumor, by mechanisms which may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, altering the physiologicfunction of PSCA, and/or the inhibition of ligand binding or signaltransduction pathways. In addition to unconjugated PSCA antibodies,fragments thereof, and recombinant proteins of the invention, PSCAantibodies conjugated to toxic agents such as ricin may also be usedtherapeutically to deliver the toxic agent directly to PSCA-bearingtumor cells and thereby destroy the tumor.

Cancer immunotherapy using PSCA antibodies may follow the teachingsgenerated from various approaches which have been successfully employedwith respect to other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit Rev Immunol 18: 133-138),multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186; Tsunenari etal., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res 52: 2771-2776), B-cell lymphoma (Funakoshi et al., 1996, JImmunther Emphasis Tumor Immunol 19: 93-101), leukemia (Zhong et al.,1996, Leuk Res 20: 581-589), colorectal cancer (Moun et al., 1994,Cancer Res 54: 6160-6166); Velders et al., 1995, Cancer Res 55:4398-4403), and breast cancer (Shepard et al., 1991, J Clin Immunol 11:117-127).

For example, one way to apply antitumor monoclonal antibodies clinicallyis to administer them in unmodified form, using monoclonal antibodies ofthe invention which display antitumor activity (e.g., ADCC and CDCactivity) and/or internalizing ability in vitro and/or in animal models(see, e.g. Hellstrom et al., Proc. Natl. Acad. Sci. USA 82:1499-1502(1985). To detect ADCC and CDC activity monoclonal antibodies can betested for lysing cultured ⁵¹Cr-labeled tumor target cells over a 4-hourincubation period. Target cells are labeled with ⁵¹Cr and then can beexposed for 4 hours to a combination of effector cells (in the form ofhuman lymphocytes purified by the use of a lymphocyte-separation medium)and antibody, which is added in concentrations, e.g., varying between0.1 μg/ml and 10 μg/ml. The release of ⁵¹Cr from the target cells ismeasured as evidence of tumor-cell lysis (cytotoxicity). Controlsinclude the incubation of target cells alone or with either lymphocytesor monoclonal antibody separately. The total amount of ⁵¹Cr that can bereleased is measured and ADCC is calculated as the percent killing oftarget cells observed with monoclonal antibody plus effector cells ascompared to target cells being incubated alone. The procedure for CDC isidentical to the one used to detect ADCC except that human serum, as asource of complement, (diluted 1:3 to 1:6) is added in place of theeffector cells.

In the practice of the method of the invention, anti-PSCA antibodiescapable of inhibiting the growth of cancer cells expressing PSCA on thecell surface are administered in a therapeutically effective amount tocancer patients whose tumors express or overexpress PSCA. The anti-PSCAmAb therapy method of the invention demonstrates remarkable tumor growthinhibition of prostate tumors in vivo. Accordingly, the inventionprovides a specific, effective and long-needed treatment for prostatecancer. The method of the invention may also be useful for the treatmentof other cancers which express or overexpress PSCA, including but notlimited to bladder carcinoma and pancreatic carcinomas, since both ofthese cancers express elevated levels of PSCA. The antibody therapymethods of the invention may be combined with a chemotherapeutic,radiation, and/or other therapeutic regimen.

As described in Example 18A below, individual mouse anti-PSCA mAbs, aswell as combinations of these anti-PSCA monoclonal antibodies, arecapable of significantly inhibiting prostate tumor growth in vivo usinga xenogenic prostate cancer SCID mouse model. In one study, a cohort ofSCID mice receiving injections of a human prostate tumor xenograft weretreated with a combination of several murine anti-PSCA mAbs. The resultsof this study showed that the treatment was able to completely block theformation of tumors in all of these mice—even after 61 days post tumorinjection. In contrast, all animals in a control group of SCID micereceiving the same prostate tumor xenograft, but treated with controlmurine IgG, developed significant and progressively more massive tumorsduring the study. There was no apparent toxicity associated with thetreatment of these animals with the anti-PSCA mAb preparation, as allmice in the treatment group remained lively and healthy throughout theexperiment. The xenograft used in the study, LAPC-9, was generated froma bone tumor biopsy of a patient with hormone-refractory metastaticprostate cancer, is characterized by an extremely androgen-sensitivephenotype (PSA levels drop to zero after castration in recipient SCIDmice), particularly aggressive growth properties, and high leveloverexpression of PSCA. LAPC-9 and is described further elsewhere(Published PCT Application WO98/16628, Sawyers et al., Apr. 23, 1998).These results were confirmed in a second in vivo study described inExample 18B. In addition, further in vivo studies demonstrated thatanti-PSCA mAbs are therapeutically effective when used alone (Example18C1, C2). In all of these in vivo studies, tumors in mice receiving theanti-PSCA mAb treatments had significantly slower growth rates, longerlatency periods, and were smaller in size compared to tumors in micereceiving control antibody treatments. Serum PSA levels were also lowerin relation to control treated animals and correlated with tumorinhibition. Moreover, antibodies recognizing different PSCA epitopes, aswell as antibodies having different IgG isotypes, are therapeuticallyeffective. In one study, anti-PSCA mAbs effectively inhibited the growthof established prostate tumors in vivo (Example 18, C4). Some of themice treated in this particular study showed tumor regression followingPSCA treatment (Example 18).

Additionally, the 3C5 antibody, administered to a tumor-bearing mouse,targeted the tumor cells that express PSCA. A SCID mouse bearing anLAPC-9 tumor (e.g., expressed PSCA), was treated with 3C5 antibody. Thetumor was explanted and examined for the presence of the 3C5 antibody,by immunohistochemistry analysis (Example 26, FIG. 71). The fixed tissueslices were probed with goat anti-mouse IgG. The 3C5 antibody waslocalized to the mass of PSCA-expressing tumor cells (FIG. 71) and couldbe detected throughout the tumor. Because SCID mice produce noimmunoglobulin, the antibody detected in the tumor tissue most likelyoriginated from the 3C5 treatment. To confirm the localization of the3C5 antibody, Western blot analysis was performed on tumor explants fromthe same mouse. The blot included protein extracts from the tumorexplant, control IgG antibody, and 3C5 antibody, and the blot was probedwith goat anti-mouse IgG-HRP antibodies. The IgG heavy and light chainswere readily detected in the tumor lysates from the 3C5-treated mouse(FIG. 72).

The results of a different study also indicate that anti-PSCA antibodiescan target PSCA-expressing tumors. A SCID mouse bearing an establishedLAPC-9 tumor was treated with 1G8 antibody. The explanted tumor wasexamined for the presence of the 1G8 antibody, by Western blot analysis(Example 26, FIG. 72) using goat anti-mouse IgG-HRP antibodies as aprobe. The heavy and light chains were readily detectable in the1G8-treated mouse. These results indicate that anti-PSCA antibodiesadministered to an subject, can circulate and target a PSCA-expressingtumor. This suggests that anti-PSCA monoclonal antibodies can circulateand target PSCA-expressing cells in tumors that are local, locallyrecurring, and metastatic. Furthermore, this suggests that conjugatedanti-PSCA monoclonal antibodies can target and kill tumors cellsexpressing PSCA.

As described in Example 24 below, individual anti-PSCA mAbs are capableof inhibiting prostate tumor growth in vivo, in a xenogenic prostatecancer SCID mouse model. For example, two cohorts of SCID mice receivedinjections of LAPC-9, and were treated with 1G8 or 3C5. The resultsshowed that treatment with 1G8 or 3C5 alone inhibited tumor growth inthe tumor-bearing mice. In contrast, the mice in a control group thatreceived the same prostate tumor xenograft, but treated with murine IgGor phosphate buffer, developed larger tumors during the study. Inaddition, the anti-PSCA treatment significantly prolonged the life ofthe mice receiving the antibody treatment, compared to the control mice.The prolonged life of the antibody-treated mice correlated with adecrease in tumor growth, and effected the level of serum PSA levels.These results indicate that treatment with anti-PSCA antibody canprolong the life of a tumor-bearing animal, by inhibiting tumor growth.

The effect of anti-PSCA mAbs in combination with an cytotoxic agent wasalso tested. As described in Example 25 below, two cohorts of SCID micereceived injections of PC3 cells which were engineered to express PSCA,and the mice were treated with 1G8 alone or in combination withdoxorubicin. The results showed that treatment with 1G8 inhibited tumorgrowth of the PSCA-positive PC3 cells, and the combination of 1G8 anddoxorubicin had a synergetic effect on inhibiting tumor growth, comparedto the tumors in mice treated with phosphate buffer or doxorubicinalone.

Thus, the results of Example 24 show that anti-PSCA monoclonalantibodies, having different isotypes, are effective in inhibiting thegrowth of established androgen-dependent tumors. For example, the LAPC-9xenograft was generated from a bone tumor biopsy of a patient withhormone-refractory metastatic prostate cancer. The 1G8 antibody is amouse gamma-1, isotypic, neutral antibody, which interacts directly withthe PSCA antigen. The 3C5 antibody is a mouse gamma-2A isotypic,antibody, which binds to cells and complement. Thus, the 1G8 antibodymay direct cell cytotoxicity of androgen-dependent tumors, through anantibody-dependent cell cytotoxicity (ADCC) mechanism, and the 3C5antibody may initiate a potent immune response against the tumor. In asimilar manner, the results of Example 25 show that anti-PSCA antibodiesare effective in treating established androgen-independent tumors.

Patients may be evaluated for the presence and level of PSCAoverexpression in tumors, preferably using immunohistochemicalassessments of tumor tissue, quantitative PSCA imaging, or othertechniques capable of reliably indicating the presence and degree ofPSCA expression. Immunohistochemical analysis of tumor biopsies orsurgical specimens may be preferred for this purpose. Methods forimmunohistochemical analysis of tumor tissues are well known in the art.An example of an immunohistochemical analytical technique useful fordetermining the level of PSCA overexpression in a sample is described inthe example sections below.

Anti-PSCA monoclonal antibodies useful in treating cancer include thosewhich are capable of initiating a potent immune response against thetumor and those which are capable of direct cytotoxicity. In thisregard, anti-PSCA mAbs may elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites or complement proteins. In addition, anti-PSCA mAbs which exert adirect biological effect on tumor growth are useful in the practice ofthe invention. Such mAbs may not require the complete immunoglobulin toexert the effect. Potential mechanisms by which such directly cytotoxicmAbs may act include inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism by which a particularanti-PSCA mAb exerts an anti-tumor effect may be evaluated using anynumber of in vitro assays designed to determine ADCC, ADMMC,complement-mediated cell lysis, and so forth, such as those described inExample 19, below.

The anti-tumor activity of a particular anti-PSCA mAb, or combination ofanti-PSCA mAbs, is preferably evaluated in vivo using a suitable animalmodel. Xenogenic cancer models, wherein human cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice, are particularly appropriate and areknown. Examples of xenograft models of human prostate cancer (capable ofrecapitulating the development of primary tumors, micrometastasis, andthe formation of osteoblastic metastases characteristic of late stagedisease) are described in Klein et al., 1997, Nature Medicine 3: 402-408and in PCT Patent Application WO98/16628, Sawyers et al., published Apr.23, 1998. The examples herein provide detailed experimental protocolsfor evaluating the anti-tumor potential of anti-PSCA mAb preparations invivo. Other in vivo assays are contemplated, such as those which measureregression of established tumors, interference with the development ofmetastasis, and the like.

It should be noted that the use of murine or other non-human monoclonalantibodies and chimeric mAbs may induce moderate to strong immuneresponses in some patients. In the most severe cases, such an immuneresponse may lead to the extensive formation of immune complexes which,potentially, can cause renal failure. Accordingly, preferred monoclonalantibodies used in the practice of the therapeutic methods of theinvention are those which are either fully human or humanized and whichbind specifically to the target PSCA antigen with high affinity butexhibit low or no antigenicity in the patient.

The method of the invention contemplate the administration of singleanti-PSCA mAbs as well as combinations, or “cocktails, of differentindividual mAbs such as those recognizing different epitopes. Such mAbcocktails may have certain advantages inasmuch as they contain mAbswhich bind to different epitopes and/or exploit different effectormechanisms or combine directly cytotoxic mAbs with mAbs that rely onimmune effector functionality. Such mAbs in combination may exhibitsynergistic therapeutic effects. In addition, the administration ofanti-PSCA mAbs may be combined with other therapeutic agents, includingbut not limited to various chemotherapeutic agents, androgen-blockers,and immune modulators (e.g., IL-2, GM-CSF). The anti-PSCA mAbs may beadministered in their “naked” or unconjugated form, or may havetherapeutic agents conjugated to them.

The anti-PSCA monoclonal antibodies used in the practice of the methodof the invention may be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material which when combined with the anti-PSCAmAbs retains the anti-tumor function of the antibody and is non-reactivewith the subject's immune systems. Examples include, but are not limitedto, any of a number of standard pharmaceutical carriers such as sterilephosphate buffered saline solutions, bacteriostatic water, and the like(see, generally, Remington's Pharmaceutical Sciences 16^(th) Edition, A.Osal., Ed., 1980).

The anti-PSCA antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. The preferred route of administration is by intravenousinjection. A preferred formulation for intravenous injection comprisesthe anti-PSCA mAbs in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. The anti-PSCA mAb preparation may be lyophilized and stored as asterile powder, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Treatment will generally involve the repeated administration of theanti-PSCA antibody preparation via an acceptable route of administrationsuch as intravenous injection (IV), at an effective dose. Dosages willdepend upon various factors generally appreciated by those of skill inthe art, including without limitation the type of cancer and theseverity, grade, or stage of the cancer, the binding affinity and halflife of the mAb or mAbs used, the degree of PSCA expression in thepatient, the extent of circulating shed PSCA antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic agents used in combination with thetreatment method of the invention. Typical daily doses may range fromabout 0.1 to 100 mg/kg. Doses in the range of 10-500 mg mAb per week maybe effective and well tolerated, although even higher weekly doses maybe appropriate and/or well tolerated. The principal determining factorin defining the appropriate dose is the amount of a particular antibodynecessary to be therapeutically effective in a particular context.Repeated administrations may be required in order to achieve tumorinhibition or regression. Initial loading doses may be higher. Theinitial loading dose may be administered as an infusion. Periodicmaintenance doses may be administered similarly, provided the initialdose is well tolerated.

Direct administration of PSCA mAbs is also possible and may haveadvantages in certain contexts. For example, for the treatment ofbladder carcinoma, PSCA mAbs may be injected directly into the bladder.Because PSCA mAbs administered directly to bladder will be cleared fromthe patient rapidly, it may be possible to use non-human or chimericantibodies effectively without significant complications ofantigenicity.

Patients may be evaluated for serum PSCA in order to assist in thedetermination of the most effective dosing regimen and related factors.The PSCA Capture ELISA described in Example 20 infra, or a similarassay, may be used for quantitating circulating PSCA levels in patientsprior to treatment. Such assays may also be used for monitoring purposesthroughout therapy, and may be useful to gauge therapeutic success incombination with evaluating other parameters such as serum PSA levels.

The invention further provides vaccines formulated to contain a PSCAprotein or fragment thereof. The use of a tumor antigen in a vaccine forgenerating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and, for example, has been employed inprostate cancer using human PSMA and rodent PAP immunogens (Hodge etal., 1995, Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol.159: 3113-3117). Such methods can be readily practiced by employing aPSCA protein, or fragment thereof, or a PSCA-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the PSCA immunogen.

For example, viral gene delivery systems may be used to deliver aPSCA-encoding nucleic acid molecule. Various viral gene delivery systemswhich can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a PSCA protein or fragment thereof introduced into the patient(e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human PSCA cDNA may be employed. In anotherembodiment, PSCA nucleic acid molecules encoding specific cytotoxic Tlymphocyte (CTL) epitopes may be employed. CTL epitopes can bedetermined using specific algorithms (e.g., Epimer, Brown University) toidentify peptides within a PSCA protein which are capable of optimallybinding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present PSCA antigen to a patient's immunesystem. Dendritic cells express MEW class I and II, B7 costimulator, andIL-12, and are thus highly specialized antigen presenting cells. Inprostate cancer, autologous dendritic cells pulsed with peptides of theprostate-specific membrane antigen (PSMA) are being used in a Phase Iclinical trial to stimulate prostate cancer patients' immune systems(Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate29: 371-380). Dendritic cells can be used to present PSCA peptides to Tcells in the context of MHC class I and II molecules. In one embodiment,autologous dendritic cells are pulsed with PSCA peptides capable ofbinding to MEW molecules. In another embodiment, dendritic cells arepulsed with the complete PSCA protein. Yet another embodiment involvesengineering the overexpression of the PSCA gene in dendritic cells usingvarious implementing vectors known in the art, such as adenovirus(Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Hendersonet al., 1996, Cancer Res. 56: 3763-3770), lentivirus, adeno-associatedvirus, DNA transfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869),and tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186: 1177-1182).

Anti-idiotypic anti-PSCA antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga PSCA protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-PSCA antibodies that mimic an epitope on aPSCA protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65-76). Such an anti-idiotypicantibody can be used in anti-idiotypic therapy as presently practicedwith other anti-idiotypic antibodies directed against tumor antigens.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing PSCA. Using the PSCA-encoding DNA moleculesdescribed herein, constructs comprising DNA encoding a PSCAprotein/immunogen and appropriate regulatory sequences may be injecteddirectly into muscle or skin of an individual, such that the cells ofthe muscle or skin take-up the construct and express the encoded PSCAprotein/immunogen. The PSCA protein/immunogen may be expressed as a cellsurface protein or be secreted. Expression of the PSCA protein/immunogenresults in the generation of prophylactic or therapeutic humoral andcellular immunity against prostate cancer. Various prophylactic andtherapeutic genetic immunization techniques known in the art may be used(for review, see information and references published at internetaddress www.genweb.com).

The invention further provides methods for inhibiting cellular activity(e.g., cell proliferation, activation, or propagation) of a cellexpressing multiple PSCA antigens on its cell surface. This methodcomprises reacting the immunoconjugates of the invention (e.g., aheterogeneous or homogenous mixture) with the cell so that the PSCAantigens on the cell surface forms a complex with the immunoconjugates.The greater the number of PSCA antigens on the cell surface, the greaterthe number of PSCA-antibody complexes can be used. The greater thenumber of PSCA-antibody complexes the greater the cellular activity thatis inhibited. A subject with a neoplastic or preneoplastic condition canbe treated in accordance with this method when the inhibition ofcellular activity results in cell death.

A heterogeneous mixture includes PSCA antibodies that recognizedifferent or the same epitope, each antibody being conjugated to thesame or different therapeutic agent. A homogenous mixture includesantibodies that recognize the same epitope, each antibody beingconjugated to the same therapeutic agent.

The invention further provides methods for inhibiting the biologicalactivity of PSCA by blocking PSCA from binding its ligand. The methodscomprises contacting an amount of PSCA with an antibody orimmunoconjugate of the invention under conditions that permit aPSCA-immunoconjugate or PSCA-antibody complex thereby blocking PSCA frombinding its ligand and inhibiting the activity of PSCA.

In another embodiment, the invention provides methods for selectivelyinhibiting a cell expressing PSCA antigen by reacting any one or acombination of the immunoconjugates of the invention with the cell in anamount sufficient to inhibit the cell. Such amounts include an amount tokill the cell or an amount sufficient to inhibit cell growth orproliferation. As discussed supra the dose and dosage regimen willdepend on the nature of the disease or disorder to be treated associatedwith PSCA, its population, the site to which the antibodies are to bedirected, the characteristics of the particular immunotoxin, and thepatient. For example, the amount of immunoconjugate can be in the rangeof 0.1 to 200 mg/kg of patient weight.

Methods for Identifying PSCA Proteins and PSCA Genes and RNA

The invention provides methods for identifying cells, tissues ororganisms expressing a PSCA protein or a PSCA gene. Such methods can beused to diagnose the presence of cells or an organism that expresses aPSCA protein in vivo or in vitro. The methods of the present inventionare particularly useful in the determining the presence of cells thatmediate pathological conditions of the prostate. Specifically, thepresence of a PSCA protein can be identified by determining whether aPSCA protein, or nucleic acid encoding a PSCA protein, is expressed. Theexpression of a PSCA protein can be used as a means for diagnosing thepresence of cells, tissues or an organism that expresses a PSCA proteinor gene.

A variety of immunological and molecular genetic techniques can be usedto determine if a PSCA protein is expressed/produced in a particularcell or sample. In general, an extract containing nucleic acid moleculesor an extract containing proteins is prepared. The extract is thenassayed to determine whether a PSCA protein, or a PSCA-encoding nucleicacid molecule, is produced in the cell.

Various polynucleotide-based detection methods well known in the art maybe employed for the detection of PSCA-encoding nucleic acid moleculesand for the detection of PSCA expressing cells in a biological specimen.For example, RT-PCR methods may be used to selectively amplify a PSCAmRNA or fragment thereof, and such methods may be employed to identifycells expressing PSCA, as described in Example 1 below. In a particularembodiment, RT-PCR is used to detect micrometastatic prostate, bladderor pancreatic cancer cells or circulating prostate, bladder orpancreatic cancer cells. Various other amplification type detectionmethods, such as, for example, branched DNA methods, and various wellknown hybridization assays using DNA or RNA probes may also be used forthe detection of PSCA-encoding polynucleotides or PSCA expressing cells.

Various methods for the detection of proteins are well known in the artand may be employed for the detection of PSCA proteins and PSCAexpressing cells. To perform a diagnostic test based on proteins, asuitable protein sample is obtained and prepared using conventionaltechniques. Protein samples can be prepared, for example, simply byboiling a sample with SDS. The extracted protein can then be analyzed todetermine the presence of a PSCA protein using known methods. Forexample, the presence of specific sized or charged variants of a proteincan be identified using mobility in an electric filed. Alternatively,antibodies can be used for detection purposes. A skilled artisan canreadily adapt known protein analytical methods to determine if a samplecontains a PSCA protein.

Alternatively, PSCA expression can also be used in methods to identifyagents that decrease the level of expression of the PSCA gene. Forexample, cells or tissues expressing a PSCA protein can be contactedwith a test agent to determine the effects of the agent on PSCAexpression. Agents that activate PSCA expression can be used as anagonist of PSCA activity whereas agents that decrease PSCA expressioncan be used as an antagonist of PSCA activity.

PSCA Promoter and Other Expression Regulatory Elements

The invention further provides expression control sequences found 5′ ofthe of the newly identified PSCA gene in a form that can be used togenerate expression vectors and transgenic animals. Specifically, thePSCA expression control elements, such as the PSCA promoter that canreadily be identified as being 5′ from the ATG start codon in the PSCAgene, and can be used to direct the expression of an operably linkedprotein encoding DNA sequence. Since PSCA expression is predominantlyexpressed in prostate cells, the expression control elements areparticularly useful in directing the expression of an introducedtransgene in a tissue specific fashion. A skilled artisan can readilyuse the PSCA gene promoter and other regulatory elements in expressionvectors using methods known in the art.

In eukaryotic cells, the regulatory sequences can be found upstream,downstream and within the coding region of the gene. The eukaryoticregulatory sequences comprise a promoter sequence and sometimes at leastone enhancer sequence. In a typical eukaryotic gene, the promotersequence resides upstream and proximal to the coding region of the gene,and must be oriented in one direction to control expression of the gene.In a typical eukaryotic gene, the enhancer sequences can reside in theupstream, downstream and even within the coding region of the gene, andcan be oriented in either direction to enhance or suppress expression ofthe gene.

The present invention provides a DNA fragment containing 9 kb ofsequences upstream of the PSCA coding region. The ability of this PSCAfragment to drive expression of an operatively linked transgene has beentested using a series of chimeric reporter constructs transfected intocells. The chimeric reporter constructs demonstrate an expressionpattern similar to that of native endogenous PSCA, and the PSCA fragmentdrives expression of the transgene when linked in the forwardorientation. Thus, this PSCA fragment comprises a PSCA upstreamregulatory region that exhibits promoter-like activity.

PSCA transcripts are also present at a significantly higher level inprostate tumor cells but not in benign prostatic hyperplasia. Thus PSCAtranscripts are detectable in a prostate-predominant manner, and aredetectable at a higher level in prostate tumor samples. Thesignificantly higher level of PSCA transcripts, or over-expression as isknown in the art, can be determined by measuring and comparing theamount of detectable PSCA transcripts in a normal prostate with aprostate tumor sample. This comparison can be performed by methods wellknown in the art, including Northern analysis and RT-PCR assays, and thedifferences in transcript levels can be quantitated. Thus, the presenceof a measurably different amount of PSCA transcripts (i.e., the numberof PSCA transcripts in the test sample exceeds the number from a normalsample) in the samples can be used to indicate the presence of prostatecancer.

PSCA expression is also observed in other human cancers, particularlybladder and pancreatic carcinomas. In the case of bladder carcinoma, thedegree of PSCA expression appears to correlate with the severity of thedisease, reaching the highest level of overexpression in invasivebladder cancer (See Example 17, below).

The pattern of PSCA transcript and protein accumulation is known, andthe PSCA upstream regulatory region has been isolated and characterized.A series of chimeric constructs comprising the PSCA upstream regulatoryregion operatively linked to a transgene has been tested. The PSCAupstream regulatory region drives expression of the transgene in variousprostate cells and cell lines, and in bladder, and to a lesser extent inkidney. Thus, the PSCA upstream region drives expression of a transgenein a prostate-predominant manner.

In preferred embodiments, DNA fragments of 9 kb, 6 kb, 3 kb, and 1 kbderived from the 5′ upstream region of the PSCA gene, as shown in FIG.42, were produced by techniques described herein. The 9 kb PSCA upstreamregion (pEGFP-PSCA) is involved with gene regulatory activity and wasdeposited on May 17, 1999 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 and has there beenidentified as follows ATCC No. PTA-80. The 9 kb fragment was obtained byamplification using a T7 primer and RIhPSCA3′-5(5′-gggaattcgcacagccttcagggtc-3′; SEQ ID NO:17).

Uses of the Fragment Having Gene Regulatory Activity

This invention provides methods (e.g., gene therapy methods) fortargeting a gene-of-interest to a cancer cell/site so that the proteinencoded by the gene can be expressed thereby directly or indirectlyameliorating the diseased state.

A susceptible cell is introduced with an expression vector thatexpresses a transgene (e.g., a therapeutic gene) under the control of aPSCA upstream region having significantly increased gene expressionactivity in tumor cells. The use of an expression vector that expressesa therapeutic gene predominantly in tumor cells will allow expression ofthe therapeutic genes in target cell, such as prostate, bladder andpancreatic tumor cells.

After infecting a susceptible cell, a transgene (e.g., a therapeuticgene) is driven by a PSCA upstream region having increased geneexpression activity in a vector, that expresses the protein encoded bythe transgene. The use of a fairly specific prostate specific genevector will allow selective expression of the specific genes in targetcells, e.g., prostate cancer cells.

PSCA regions having increased gene expression activity may be modified,e.g., by sequence mutations, deletions, and insertions, so as to producederivative molecules. Modifications include multiplying the number ofsequences that can bind prostate cell specific regulatory proteins anddeleting sequences that are nonfunctional in the PSCA region having geneexpression activity. Other modifications include adding enhancersthereby improving the efficiency of the PSCA region having promoteractivity. Enhancers may function in a position-independent manner andcan be located upstream, within or downstream of the transcribed region.

Derivative molecules would retain the functional property of the PSCAupstream region having increased gene expression activity, namely, themolecule having such substitutions will still permit substantiallyprostate tissue specific expression of a gene of interest located 3′ tothe fragment. Modification is permitted so long as the derivativemolecules retain its ability to drive gene expression in a substantiallyprostate specific manner compared to a PSCA fragment having promoteractivity alone.

In a preferred embodiment, a vector was constructed by inserting aheterologous sequence (therapeutic gene) downstream of the PSCA upstreamregion having promoter activity.

Examples of therapeutic genes include suicide genes. These are genessequences the expression of which produces a protein or agent thatinhibits tumor cell growth or tumor cell death (e.g., prostate tumorcells). Suicide genes include genes encoding enzymes (e.g., prodrugenzymes), oncogenes, tumor suppressor genes, genes encoding toxins,genes encoding cytokines, or a gene encoding oncostatin. The purpose ofthe therapeutic gene is to inhibit the growth of or kill the cancer cellor produce cytokines or other cytotoxic agents which directly orindirectly inhibit the growth of or kill the cancer cells.

Suitable prodrug enzymes include thymidine kinase (TK), xanthine-guaninephosphoribosyltransferase (GPT) gene from E. Coli or E. Coli cytosinedeaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT).

Suitable oncogenes and tumor suppressor genes include neu, EGF, ras(including H, K, and N ras), p53, Retinoblastoma tumor suppressor gene(Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase (PTPase),and nm23. Suitable toxins include Pseudomonas exotoxin A and S;diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-like toxins(SLT-1, -2), ricin, abrin, supporin, and gelonin.

Suitable cytokines include interferons, GM-CSF interleukins, tumornecrosis factor (TNF) (Wong G, et al., Human GM-CSF: Molecular cloningof the complementary DNA and purification of the natural and recombinantproteins. Science 1985; 228:810); WO9323034 (1993); Horisberger M A, etal., Cloning and sequence analyses of cDNAs for interferon- andvirus-induced human Mx proteins reveal that they contain putativeguanine nucleotide-binding sites: functional study of the correspondinggene promoter. Journal of Virology, 1990 Mar., 64(3):1171-81; Li Y P etal., Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, butnot IL-1, down-regulate the osteocalcin gene promoter. Journal ofImmunology, 1992 Feb. 1, 148(3):788-94; Pizarro T T, et al. Induction ofTNF alpha and TNF beta gene expression in rat cardiac transplants duringallograft rejection. Transplantation, 1993 Aug., 56(2):399-404).(Breviario F, et al., Interleukin-1-inducible genes in endothelialcells. Cloning of a new gene related to C-reactive protein and serumamyloid P component. Journal of Biological Chemistry, 1992 Nov. 5,267(31):22190-7; Espinoza-Delgado I, et al., Regulation of IL-2 receptorsubunit genes in human monocytes. Differential effects of IL-2 andIFN-gamma. Journal of Immunology, 1992 Nov. 1, 149(9):2961-8; Algate PA, et al., Regulation of the interleukin-3 (IL-3) receptor by IL-3 inthe fetal liver-derived FL5.12 cell line. Blood, 1994 May 1,83(9):2459-68; Cluitmans F H, et al., IL-4 down-regulates IL-2-, IL-3-,and GM-CSF-induced cytokine gene expression in peripheral bloodmonocytes. Annals of Hematology, 1994 Jun., 68(6):293-8; Lagoo, A S, etal., IL-2, IL-4, and IFN-gamma gene expression versus secretion insuperantigen-activated T cells. Distinct requirement for costimulatorysignals through adhesion molecules. Journal of Immunology, 1994 Feb. 15,152(4):1641-52; Martinez O M, et al., IL-2 and IL-5 gene expression inresponse to alloantigen in liver allograft recipients and in vitro.Transplantation, 1993 May, 55(5):1159-66; Pang G, et al., GM-CSF, IL-1alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expressionand cytokine production in human duodenal fibroblasts stimulated withlipopolysaccharide, IL-1 alpha and TNF-alpha. Clinical and ExperimentalImmunology, 1994 Jun., 96(3):437-43; Ulich T R, et al.,Endotoxin-induced cytokine gene expression in vivo. III. IL-6 mRNA andserum protein expression and the in vivo hematologic effects of IL-6.Journal of Immunology, 1991 Apr. 1, 146(7):2316-23; Mauviel A, et al.,Leukoregulin, a T cell-derived cytokine, induces IL-8 gene expressionand secretion in human skin fibroblasts. Demonstration and secretion inhuman skin fibroblasts. Demonstration of enhanced NF-kappa B binding andNF-kappa B-driven promoter activity. Journal of Immunology, 1992 Nov. 1,149(9):2969-76).

Growth factors include Transforming Growth Factor-α (TGFα) and β (TGFβ),cytokine colony stimulating factors (Shimane M, et al., Molecularcloning and characterization of G-CSF induced gene cDNA. Biochemical andBiophysical Research Communications, 1994 Feb. 28, 199(1):26-32; Kay AB, et al., Messenger RNA expression of the cytokine gene cluster,interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophagecolony-stimulating factor, in allergen-induced late-phase cutaneousreactions in atopic subjects. Journal of Experimental Medicine, 1991Mar. 1, 173(3):775-8; de Wit H, et al., Differential regulation of M-CSFand IL-6 gene expression in monocytic cells. British Journal ofHaematology, 1994 Feb., 86(2):259-64; Sprecher E, et al., Detection ofIL-1 beta, TNF-alpha, and IL-6 gene transcription by the polymerasechain reaction in keratinocytes, Langerhans cells and peritoneal exudatecells during infection with herpes simplex virus-1. Archives ofVirology, 1992, 126(1-4):253-69).

Vectors suitable for use in the methods of the present invention areviral vectors including adenoviruses, lentivirus, retroviral vectors,adeno-associated viral (AAV) vectors.

Preferably, the viral vector selected should meet the followingcriteria: 1) the vector must be able to infect the tumor cells and thusviral vectors having an appropriate host range must be selected; 2) thetransferred gene should be capable of persisting and being expressed ina cell for an extended period of time; and 3) the vector should be safeto the host and cause minimal cell transformation. Retroviral vectorsand adenoviruses offer an efficient, useful, and presently thebest-characterized means of introducing and expressing foreign genesefficiently in mammalian cells. These vectors have very broad host andcell type ranges, express genes stably and efficiently. The safety ofthese vectors has been proved by many research groups. In fact many arein clinical trials.

Other virus vectors that may be used for gene transfer into cells forcorrection of disorders include retroviruses such as Moloney murineleukemia virus (MoMuLV); papovaviruses such as JC, SV40, polyoma;Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovine papilloma virustype I (BPV); vaccinia and poliovirus and other human and animalviruses.

Adenoviruses have several properties that make them attractive ascloning vehicles (Bachettis et al.: Transfer of gene for thymidinekinase-deficient human cells by purified herpes simplex viral DNA. PNASUSA, 1977 74:1590; Berkner, K. L.: Development of adenovirus vectors forexpression of heterologous genes. Biotechniques, 1988 6:616;Ghosh-Choudhury G, et al., Human adenovirus cloning vectors based oninfectious bacterial plasmids. Gene 1986; 50:161; Hag-Ahmand Y, et al.,Development of a helper-independent human adenovirus vector and its usein the transfer of the herpes simplex virus thymidine kinase gene. JVirol 1986; 57:257; Rosenfeld M, et al., Adenovirus-mediated transfer ofa recombinant α₁-antitrypsin gene to the lung epithelium in vivo.Science 1991; 252:431).

For example, adenoviruses possess an intermediate sized genome thatreplicates in cellular nuclei; many serotypes are clinically innocuous;adenovirus genomes appear to be stable despite insertion of foreigngenes; foreign genes appear to be maintained without loss orrearrangement; and adenoviruses can be used as high level transientexpression vectors with an expression period up to 4 weeks to severalmonths. Extensive biochemical and genetic studies suggest that it ispossible to substitute up to 7-7.5 kb of heterologous sequences fornative adenovirus sequences generating viable, conditional,helper-independent vectors (Kaufman R. J.; identification of thecomponent necessary for adenovirus translational control and theirutilization in cDNA expression vectors. PNAS USA, 1985 82:689).

AAV is a small human parvovirus with a single stranded DNA genome ofapproximately 5 kb. This virus can be propagated as an integratedprovirus in several human cell types. AAV vectors have several advantagefor human gene therapy. For example, they are trophic for human cellsbut can also infect other mammalian cells; (2) no disease has beenassociated with AAV in humans or other animals; (3) integrated AAVgenomes appear stable in their host cells; (4) there is no evidence thatintegration of AAV alters expression of host genes or promoters orpromotes their rearrangement; (5) introduce genes can be rescued fromthe host cell by infection with a helper virus such as adenovirus.

HSV-1 vector system facilitates introduction of virtually any gene intonon-mitotic cells (Geller et al. an efficient deletion mutant packagingsystem for a defective herpes simplex virus vectors: Potentialapplications to human gene therapy and neuronal physiology. PNAS USA,1990 87:8950).

Another vector for mammalian gene transfer is the bovine papillomavirus-based vector (Sarver N, et al., Bovine papilloma virus DNA: Anovel eukaryotic cloning vector. Mol Cell Biol 1981; 1:486).

Vaccinia and other poxvirus-based vectors provide a mammalian genetransfer system. Vaccinia virus is a large double-stranded DNA virus of120 kilodaltons (kd) genomic size (Panicali D, et al., Construction ofpoxvirus as cloning vectors: Insertion of the thymidine kinase gene fromherpes simplex virus into the DNA of infectious vaccine virus. Proc NatlAcad Sci USA 1982; 79:4927; Smith et al. infectious vaccinia virusrecombinants that express hepatitis B virus surface antigens. Nature,1983 302:490.)

Retroviruses are packages designed to insert viral genes into host cells(Guild B, et al., Development of retrovirus vectors useful forexpressing genes in cultured murine embryonic cells and hematopoieticcells in vivo. J Virol 1988; 62:795; Hock R A, et al., Retrovirusmediated transfer and expression of drug resistance genes in humanhemopoietic progenitor cells. Nature 1986; 320:275).

The basic retrovirus consists of two identical strands of RNA packagedin a proviral protein. The core surrounded by a protective coat calledthe envelope, which is derived from the membrane of the previous hostbut modified with glycoproteins contributed by the virus.

Preferably, for treating defects, disease or damage of cells in, forexample, the prostate, vectors of the invention include a therapeuticgene or transgenes, for example a gene encoding TK. The geneticallymodified vectors are administered into the prostate to treat defects,disease such as prostate cancer by introducing a therapeutic geneproduct or products into the prostate that enhance the production ofendogenous molecules that have ameliorative effects in vivo. The sameprinciples apply with respect to the treatment of other cancers, such aspancreatic, bladder or other cancers expressing PSCA.

The basic tasks in this embodiment of the present method of theinvention are isolating the gene of interest, attaching it to a fragmenthaving gene regulatory activity, selecting the proper vector vehicle todeliver the gene of interest to the body, administering the vectorhaving the gene of interest into the body, and achieving appropriateexpression of the gene of interest to a target cell. The presentinvention provides packaging the cloned genes, i.e. the genes ofinterest, in such a way that they can be injected directly into thebloodstream or relevant organs of patients who need them. The packagingwill protect the foreign DNA from elimination by the immune system anddirect it to appropriate tissues or cells.

Along with the human or animal gene of interest another gene, e.g., aselectable marker, can be inserted that will allow easy identificationof cells that have incorporated the modified retrovirus. The criticalfocus on the process of gene therapy is that the new gene must beexpressed in target cells at an appropriate level with a satisfactoryduration of expression.

The methods described below to modify vectors and administering suchmodified vectors into the target organ (e.g., prostate) are merely forpurposes of illustration and are typical of those that might be used.However, other procedures may also be employed, as is understood in theart.

Most of the techniques used to construct vectors and the like are widelypracticed in the art, and most practitioners are familiar with thestandard resource materials which describe specific conditions andprocedures. However, for convenience, the following paragraphs may serveas a guideline.

General Methods for Vector Construction

Construction of suitable vectors containing the desired therapeutic genecoding and control sequences employs standard ligation and restrictiontechniques, which are well understood in the art (see Maniatis et al.,in Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1982)). Isolated plasmids, DNA sequences, orsynthesized oligonucleotides are cleaved, tailored, and religated in theform desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes (See,e.g. New England Biolabs Product Catalog). In general, about 1 μg ofplasmid or DNA sequences is cleaved by one unit of enzyme in about 20 μlof buffer solution. Typically, an excess of restriction enzyme is usedto insure complete digestion of the DNA substrate.

Incubation times of about one hour to two hours at about 37° C. areworkable, although variations can be tolerated. After each incubation,protein is removed by extraction with phenol/chloroform, and may befollowed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° C. to 25° C. in 50 mM Tris (pH 7.6) 50 mMNaCl, 6 mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fillsin at 5′ sticky ends but chews back protruding 3′ single strands, eventhough the four dNTPs are present. If desired, selective repair can beperformed by supplying only one of the dNTPs, or with selected dNTPs,within the limitations dictated by the nature of the sticky ends. Aftertreatment with Klenow, the mixture is extracted with phenol/chloroformand ethanol precipitated. Treatment under appropriate conditions with Slnuclease or Bal-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 10-50 μl volumes under the following standardconditions and temperatures using T4 DNA ligase. Ligation protocols arestandard (D. Goeddel (ed.) Gene Expression Technology: Methods inEnzymology (1991)).

In vector construction employing “vector fragments”, the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) or calfintestinal alkaline phosphatase (CIP) in order to remove the 5′phosphate and prevent religation of the vector. Alternatively,religation can be prevented in vectors which have been double digestedby additional restriction enzyme digestion of the unwanted fragments.

Suitable vectors include viral vector systems e.g. ADV, RV, and AAV (R.J. Kaufman “Vectors used for expression in mammalian cells” in GeneExpression Technology, edited by D. V. Goeddel (1991).

Many methods for inserting functional DNA transgenes into cells areknown in the art. For example, non-vector methods include nonviralphysical transfection of DNA into cells; for example, microinjection(DePamphilis et al., BioTechnique 6:662-680 (1988)); liposomal mediatedtransfection (Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417(1987), Feigner and Holm, Focus 11:21-25 (1989) and Feigner et al.,Proc. West. Pharmacol. Soc. 32: 115-121 (1989)) and other methods knownin the art.

Administration of Modified Vectors into Subject

One way to get DNA into a target cell is to put it inside a membranebound sac or vesicle such as a spheroplast or liposome, or by calciumphosphate precipitation (CaPO₄) (Graham F. and Van der Eb, A., Virology52:456 1973; Schaefer-Ridder M., et al., Liposomes as gene carriers:Efficient transduction of mouse L cells by thymidine kinase gene.Science 1982; 215:166; Stavridis J C, et al., Construction oftransferrin-coated liposomes for in vivo transport of exogenous DNA tobone marrow erythroblasts in rabbits. Exp Cell Res 1986; 164:568-572).

A vesicle can be constructed in such a way that its membrane will fusewith the outer membrane of a target cell. The vector of the invention invesicles can home into the target cells. The spheroplasts are maintainedin high ionic strength buffer until they can be fused through themammalian target cell using fusogens such as polyethylene glycol.

Liposomes are artificial phospholipid vesicles. Vesicles range in sizefrom 0.2 to 4.0 micrometers and can entrap 10% to 40% of an aqueousbuffer containing macromolecules. The liposomes protect the DNA fromnucleases and facilitate its introduction into target cells.Transfection can also occur through electroporation.

Before administration, the modified vectors are suspended in completePBS at a selected density for injection. In addition to PBS, anyosmotically balanced solution which is physiologically compatible withthe subject may be used to suspend and inject the modified vectors intothe host.

For injection, the cell suspension is drawn up into the syringe andadministered to anesthetized recipients. Multiple injections may be madeusing this procedure. The viral suspension procedure thus permitsadministration of genetically modified vectors to any predetermined sitein the target organ (e.g., prostate), is relatively non-traumatic,allows multiple administrations simultaneously in several differentsites or the same site using the same viral suspension. Multipleinjections may consist of a mixture of therapeutic genes.

Uses of the Modified Vectors

The present invention provides methods for maintaining and increasingexpression of therapeutic genes using a fragment having expressionactivity.

The methods of the invention are exemplified by embodiments in whichmodified vectors carrying a therapeutic gene are injected into asubject.

In a first embodiment a protein product is expressed comprising growingthe host vector system of the invention so as to produce the protein inthe host and recovering the protein so produced. This method permits theexpression of genes of interest in both unicellular and multicellularorganisms. For example, in an in vitro assay, prostate cells having thevector of the invention comprising a gene of interest (e.g., the rasgene) may be used in microtiter wells as an unlimited for the ras geneproduct. A sample from a subject would be added to the wells to detectthe presence of antibodies directed against the ras gene. This assay canaid in the quantitative and qualitative determination of the presence ofras antibodies in the sample for the clinical assessment of whether thesubject's immune system is combating the disease associated withelevated levels of ras.

In a second embodiment metastatic prostate cancer is treated via genetherapy, i.e., the correction of a disease phenotype in vivo through theuse of the nucleic acid molecules of the invention.

In accordance with the practice of this invention, the subject of thegene therapy may be a human, equine, porcine, bovine, murine, canine,feline, or avian subject. Other mammals are also included in thisinvention.

The most effective mode of administration and dosage regimen for themolecules of the present invention depends upon the exact location ofthe prostate tumor being treated, the severity and course of the cancer,the subject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject. The molecules may be delivereddirectly or indirectly via another cell, autologous cells are preferred,but heterologous cells are encompassed within the scope of theinvention.

The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m² of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided dose may beadministered daily or proportionally reduced depending on the specifictherapeutic situation).

It would be clear that the dose of the molecules of the inventionrequired to achieve treatment may be further modified with scheduleoptimization.

Generation of Transgenic Animals

Another aspect of the invention provides transgenic non-human mammalscomprising PSCA nucleic acids. For example, in one application,PSCA-deficient non-human animals can be generated using standardknock-out procedures to inactivate a PSCA homologue or, if such animalsare non-viable, inducible PSCA homologue antisense molecules can be usedto regulate PSCA homologue activity/expression. Alternatively, an animalcan be altered so as to contain a human PSCA-encoding nucleic acidmolecule or an antisense-PSCA expression unit that directs theexpression of PSCA protein or the antisense molecule in a tissuespecific fashion. In such uses, a non-human mammal, for example a mouseor a rat, is generated in which the expression of the PSCA homologuegene is altered by inactivation or activation and/or replaced by a humanPSCA gene. This can be accomplished using a variety of art-knownprocedures such as targeted recombination. Once generated, the PSCAhomologue deficient animal, the animal that expresses PSCA (human orhomologue) in a tissue specific manner, or an animal that expresses anantisense molecule can be used to (1) identify biological andpathological processes mediated by the PSCA protein, (2) identifyproteins and other genes that interact with the PSCA proteins, (3)identify agents that can be exogenously supplied to overcome a PSCAprotein deficiency and (4) serve as an appropriate screen foridentifying mutations within the PSCA gene that increase or decreaseactivity.

For example, it is possible to generate transgenic mice expressing thehuman minigene encoding PSCA in a tissue specific-fashion and test theeffect of over-expression of the protein in tissues and cells thatnormally do not contain the PSCA protein. This strategy has beensuccessfully used for other genes, namely bcl-2 (Veis et al. Cell 199375:229). Such an approach can readily be applied to the PSCAprotein/gene and can be used to address the issue of a potentialbeneficial or detrimental effect of the PSCA proteins in a specifictissue.

Further, in another embodiment, the invention provides a transgenicanimal having germ and somatic cells comprising an oncogene which islinked to a PSCA upstream region effective for the expression of saidgene in the tissues of said mouse for the promotion of a cancerassociated with the oncogene, thereby producing a mouse model of thatcancer.

Compositions

The invention provides a pharmaceutical composition comprising a PSCAnucleic acid molecule of the invention or an expression vector encodinga PSCA protein or encoding a fragment thereof and, optionally, asuitable carrier. The invention additionally provides a pharmaceuticalcomposition comprising an antibody or fragment thereof which recognizesand binds a PSCA protein. In one embodiment, the antibody or fragmentthereof is conjugated or linked to a therapeutic drug or a cytotoxicagent.

Suitable carriers for pharmaceutical compositions include any materialwhich when combined with the nucleic acid or other molecule of theinvention retains the molecule's activity and is non-reactive with thesubject's immune systems. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets including coated tablets and capsules. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well known conventional methods. Such compositions may also beformulated within various lipid compositions, such as, for example,liposomes as well as in various polymeric compositions, such as polymermicrospheres.

The invention also provides a diagnostic composition comprising a PSCAnucleic acid molecule, a probe that specifically hybridizes to a nucleicacid molecule of the invention or to any part thereof, or a PSCAantibody or fragment thereof. The nucleic acid molecule, the probe orthe antibody or fragment thereof can be labeled with a detectablemarker. Examples of a detectable marker include, but are not limited to,a radioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme. Further, theinvention provides a diagnostic composition comprising a PSCA-specificprimer pair capable of amplifying PSCA-encoding sequences usingpolymerase chain reaction methodologies, such as RT-PCR.

EXAMPLES Example 1 Identification and Molecular Characterization of aNovel Prostate Cell Surface Antigen (PSCA) Materials and Methods

LAPC-4 Xenografts:

LAPC-4 xenografts were generated as described in Klein et al, 1997,Nature Med. 3: 402-408.

RDA, Northern Analysis and RT-PCR:

Representational difference analysis of androgen dependent andindependent LAPC-4 tumors was performed as previously described (Braunet al., 1995, Mol. Cell. Biol. 15: 4623-4630). Total RNA was isolatedusing Ultraspec^(R) RNA isolation systems (Biotecx, Houston, Tex.)according to the manufacturer's instructions. Northern filters wereprobed with a 660 bp RDA fragment corresponding to the coding sequenceand part of the 3′ untranslated sequence of PSCA or a ˜400 bp fragmentof PSA. The human multiple tissue blot was obtained from Clontech andprobed as specified. For reverse transcriptase (RT)-PCR analysis, firststrand cDNA was synthesized from total RNA using the GeneAmp RNA PCRcore kit (Perkin Elmer-Roche, New Jersey). For RT-PCR of human PSCAtranscripts, primers 5′ primer -tgcttgccctgttgatggcag- (SEQ ID NO:12)and 3′ primer -ccagagcagcaggccgagtgca- (SEQ ID NO:13) were used toamplify a ˜320 bp fragment. Thermal cycling was performed by 25-25cycles of 95° for 30 sec, 60° for 30 sec and 72° for 1 min, followed byextension at 72° for 10 min. Primers for GAPDH (Clontech) were used ascontrols. For mouse PSCA, the primers used were 5′ primer-ttctcctgctggccacctac- (SEQ ID NO:7) and 3′ primer-gcagctcatcccttcacaat- (SEQ ID NO:8).

In Situ Hybridization Assay for PSCA mRNA:

For mRNA in situ hybridization, recombinant plasmid pCR II (1 ug,Invitrogen, San Diego, Calif.) containing the full-length PSCA gene waslinearized to generate sense and antisense digoxigenin labeledriboprobes. In situ hybridization was performed on an automatedinstrument (Ventana Gen II, Ventana Medical Systems) as previouslydescribed (Magi-Galluzzi et al., 1997, Lab. Invest. 76: 37-43). Prostatespecimens were obtained from a previously described database which hasbeen expanded to ˜130 specimens (Magi-Galluzzi et al., supra). Slideswere read and scored by two pathologists in a blinded fashion. Scores of0-3 were assigned according to the percentage of positive cells (0=0%;1=<25%; 2=25-50%; 3=>50%) and the intensity of staining (0=0; 1=1+;2=2+; 3=3+). The two scores were multiplied to give an overall score of0-9.

Results

Human PSCA cDNA:

Representational Difference Analysis (RDA), a PCR-based subtractivehybridization technique, was used to compare gene expression betweenhormone dependent and hormone independent variants of a human prostatecancer xenograft (LAPC-4) and to isolate cDNAs upregulated in theandrogen-independent LAPC-4 subline. Multiple genes were cloned,sequenced, and checked for differential expression. One 660 bp fragment(clone #15) was identified which was found to be highly overexpressed inxenograft tumors when compared with normal prostate. Comparison of theexpression of this clone to that of PSA in normal prostate and xenografttumors suggested that clone #15 was relatively cancer specific (FIG.9A).

Sequence analysis revealed that clone #15 had no exact match in thedatabases, but shared 30% nucleotide homology with stem cell antigen 2,a member of the Thy-1/Ly-6 superfamily of glycosylphosphatidylinositol(GPI)-anchored cell surface antigens. Clone #15 encodes a 123 amino acidprotein which is 30% identical to SCA-2 (also called RIG-E) and containsa number of highly conserved cysteine residues characteristic of theLy-6/Thy-1 gene family (FIG. 3). Consistent with its homology to afamily of GPI-anchored proteins, clone #15 contains both anamino-terminal hydrophobic signal sequence and a carboxyl-terminalstretch of hydrophobic amino acids preceded by a group of small aminoacids defining a cleavage/binding site for GPI linkage (Udenfriend andKodukula, 1995, Ann. Rev. Biochem. 64: 563-591). It also contains fourpredicted N-glycosylation sites. Because of its strong homology to thestem cell antigen-2, clone #15 was renamed prostate stem cell antigen(PSCA). 5′ and 3′ PCR RACE analysis was then performed using cDNAobtained from the LAPC-4 androgen independent xenograft and the fulllength cDNA nucleotide sequence (including the coding and untranslatedregions) was obtained. The nucleotide sequence of the full length cDNAencoding human PSCA is shown in FIG. 1A and the translated amino acidsequence is shown in FIG. 1B and in FIG. 3.

PSCA is Expressed in Prostate Cells:

The distribution of PSCA mRNA in normal human tissues was examined byNorthern blot analysis. The results, shown in FIG. 9B, demonstrate thatPSCA is expressed predominantly in prostate, with a lower level ofexpression present in placenta. Small amounts of mRNA can be detected inkidney and small intestine after prolonged exposure and at approximately1/100th of the level seen in prostate tissue. RT-PCR analysis of PSCAexpression in normal human tissues also demonstrates that PSCAexpression is restricted. In a panel of normal tissues, high level PSCAmRNA expression was detected in prostate, with significant expressiondetected in placenta and tonsils (FIG. 7A). RT-PCR analysis of PSCA mRNAexpression in a variety of prostate cancer xenografts prostate cancercell lines and other cell lines, and normal prostate showed high levelexpression restricted to normal prostate, the LAPC-4 and LAPC-9 prostatecancer xenografts, and the ovarian cancer cell line A431 (FIG. 7B). Themajor PSCA transcript in normal prostate is ˜1 kb (FIG. 9B). Mouse PSCAexpression was analyzed by RT-PCR in mouse spleen, liver, lung,prostate, kidney and testis. Like human PSCA, murine PSCA is expressedpredominantly in prostate. Expression can also be detected in kidney ata level similar to that seen for placenta in human tissues.

The expression of PSCA, PSMA and PSA in prostate cancer cell lines andxenografts was compared by Northern blot analysis. The results shown inFIG. 10A-C demonstrate high level prostate cancer specific expression ofboth PSCA and PSMA, whereas PSA expression is not prostate cancerspecific.

PSCA is Expressed by a Subset of Basal Cells in Normal Prostate:

Normal prostate contains two major epithelial cell populations—secretoryluminal cells and subjacent basal cells. In situ hybridizations wereperformed on multiple sections of normal prostate using an antisenseriboprobe specific for PSCA to localize its expression. As shown in FIG.11A-C, PSCA is expressed exclusively in a subset of normal basal cells.Little to no staining is seen in stroma, secretory cells or infiltratinglymphocytes. Hybridization with sense PSCA riboprobes showed nobackground staining. Hybridization with an antisense probe for GAPDHconfirmed that the RNA in all cell types was intact. Because basal cellsrepresent the putative progenitor cells for the terminallydifferentiated secretory cells, these results suggest that PSCA may be aprostate-specific stem/progenitor cell marker (Bonkhoff et al., 1994,Prostate 24: 114-118). In addition, since basal cells areandrogen-independent, the association of PSCA with basal cells raisesthe possibility that PSCA may play a role in androgen-independentprostate cancer progression.

PSCA is Overexpressed in Prostate Cancer Cells:

The initial analysis comparing PSCA expression in normal prostate andLAPC-4 xenograft tumors suggested that PSCA was overexpressed inprostate cancer. As demonstrated by the Northern blot analysis as shownin FIG. 9A-B, LAPC-4 prostate cancer tumors strongly express PSCA;however, there is almost no detectable expression in sample of BPH. Incontrast, PSA expression is clearly detectable in normal prostate, atlevels 2-3 times those seen in the LAPC-4 tumors. Thus, the expressionof PSCA in prostate cancer appears to be the reverse of what is seenwith PSA. While PSA is expressed more strongly in normal than malignantprostate tissue, PSCA is expressed more highly in prostate cancer.

To confirm the PSCA expression and its value in diagnosing prostatecancer, one hundred twenty six paraffin-embedded prostate cancerspecimens were analyzed by mRNA in situ hybridization for PSCAexpression. Specimens were obtained from primary tumors removed byradical prostatectomy or transurethral resection in all cases exceptone. All specimens were probed with both a sense and antisense constructin order to control for background staining. Slides were assigned acomposite score as describe under Materials and Methods, with a score of6 to 9 indicating strong expression and a score of 4 meaning moderateexpression. 102/126 (81%) of cancers stained strongly for PSCA, whileanother 9/126 (7%) displayed moderate staining (FIGS. 11B and 11C). Highgrade prostatic intraepithelial neoplasia, the putative precursor lesionof invasive prostate cancer, stained strongly positive for PSCA in 82%(97/118) of specimens (FIG. 11B) (Yang et al., 1997, Am. J. Path. 150:693-703). Normal glands stained consistently weaker than malignantglands (FIG. 11B). Nine specimens were obtained from patients treatedprior to surgery with hormone ablation therapy. Seven of nine (78%) ofthese residual presumably androgen-independent cancers overexpressedPSCA, a percentage similar to that seen in untreated cancers. Becausesuch a large percentage of specimens expressed PSCA mRNA, no statisticalcorrelation could be made between PSCA expression and pathologicalfeatures such as tumor stage and grade. These results suggest that PSCAmRNA overexpression is a common feature of androgen-dependent andindependent prostate cancer.

PSCA is Expressed in Androgen Independent Prostate Cancer Cell Lines:

Although PSCA was initially cloned using subtractive hybridization,Northern blot analysis demonstrated strong PSCA expression in bothandrogen-dependent and androgen-independent LAPC-4 xenograft tumors(FIG. 9A). Moreover, PSCA expression was detected in all prostate cancerxenografts, including the LAPC-4 and LAPC-9 xenografts.

PSCA expression in the androgen-independent, androgen receptor-negativeprostate cancer cell lines PC3 and DU145 was also detected byreverse-transcriptase polymerase chain reaction analysis. These datasuggest that PSCA can be expressed in the absence of functional androgenreceptor.

Example 2 Biochemical Characterization of PSCA

This experiment shows that PSCA is a glycosylated, GPI-anchored cellsurface protein.

Materials and Methods

Polyclonal Antibodies and Immunoprecipitations:

Rabbit polyclonal antiserum was generated against the synthetic peptide-TARIRAVGLLTVISK- (SEQ ID NO:9) and affinity purified using aPSCA-glutathione S transferase fusion protein. 293T cells weretransiently transfected with pCDNA II (Invitrogen, San Diego, Calif.)expression vectors containing PSCA, CD59, E25 or vector alone by calciumphosphate precipitation. Immunoprecipitation was performed as previouslydescribed (Harlow and Lane, 1988, Antibodies: A Laboratory Manual. (ColdSpring Harbor Press)). Briefly, cells were labeled with 500 uCi oftrans35S label (ICN, Irvine, Calif.) for six hours. Cell lysates andconditioned media were incubated with 1 ug of purified rabbit anti-PSCAantibody and 20 ul protein A sepharose CL-4B (Pharmacia Biotech, Sweden)for two hours. For deglycosylation, immunoprecipitates were treatedovernight at 37° with 1 u N-glycosidase F (Boehringer Mannheim) or 0.1 uneuraminidase (Sigma, St. Louis, Mo.) for 1 hour followed by overnightin 2.5 mU O-glycosidase (Boehringer Mannheim).

Flow Cytometry:

For flow cytometric analysis of PSCA cell surface expression, singlecell suspensions were stained with 2 ug/ml of purified anti-PSCAantibody and a 1:500 dilution of fluorescein isothiocyanate (FITC)labeled anti-rabbit IgG (Jackson Laboratories, West Grove, Pa.). Datawas acquired on a FACScan (Becton Dickinson) and analyzed using LYSIS IIsoftware. Control samples were stained with secondary antibody alone.Glycosylphosphatidyl inositol linkage was analyzed by digestion of 2×10⁶cells with 0.5 units of phosphatidylinositol-specific phospholipase C(PI-PLC, Boehringer Mannheim) for 90 min at 37° C. Cells were analyzedprior to and after digestion by either FACS scanning or immunoblotting.

Results

PSCA is a GPI-Anchored Glycoprotein Expressed on the Cell Surface:

The deduced PSCA amino acid sequence predicts that PSCA is heavilyglycosylated and anchored to the cell surface through a GPI mechanism.In order to test these predictions, we produced an affinity purifiedpolyclonal antibody raised against a unique PSCA peptide (see Materialsand Methods). This peptide contains no glycosylation sites and waspredicted, based on comparison to the three dimensional structure ofCD59 (another GPI-anchored PSCA homologue), to lie in an exposed portionof the mature protein (Kiefer et al., 1994, Biochem. 33: 4471-4482).Recognition of PSCA by the affinity-purified antibody was demonstratedby immunoblot and immunoprecipitation analysis of extracts of 293T cellstransfected with PSCA and a GST-PSCA fusion protein. The polyclonalantibody immunoprecipitates predominantly a 24 kd band fromPSCA-transfected, but not mock-transfected cells (FIG. 12A). Threesmaller bands are also present, the smallest being ˜10 kd. Theimmunoprecipitate was treated with N and O specific glycosidases inorder to determine if these bands represented glycosylated forms ofPSCA. N-glycosidase F deglycosylated PSCA, whereas O-glycosidase had noeffect (FIG. 12A). Some GPI-anchored proteins are known to have bothmembrane-bound and secreted forms (Fritz and Lowe, 1996, Am. J. Physiol.270: G176-G183). FIG. 12B indicates that some PSCA is secreted in the293T-overexpressing system. The secreted form of PSCA migrates at alower molecular weight than the cell surface-associated form, perhapsreflecting the absence of the covalent GPI-linkage. This result mayreflect the high level of expression in the 293T cell line and needs tobe confirmed in prostate cancer cell lines and in vivo.

Fluorescence activated cell sorting (FACS) analysis was used to localizePSCA expression to the cell surface. Nonpermeabilized mock-transfected293T cells, PSCA-expressing 293T cells and LAPC-4 cells were stainedwith affinity purified antibody or secondary antibody alone. FIG. 12Cshows cell surface expression of PSCA in PSCA-transfected 293T andLAPC-4 cells, but not in mock-transfected cells. To confirm that thiscell surface expression is mediated by a covalent GPI-linkage, cellswere treated with GPI-specific phospholipase C (PLC). Release of PSCAfrom the cell surface by PLC was indicated by a greater than one logreduction in fluorescence intensity. Recovery of PSCA in post digestconditioned medium was also confirmed by immunoblotting. The specificityof phospholipase C digestion for GPI-anchored proteins was confirmed byperforming the same experiment on 293T cells transfected with theGPI-linked antigen CD59 or the non-GPI linked transmembrane protein E25a(Deleersnijder et al., 1996, J. Biol. Chem 271: 19475-19482). PLCdigestion reduced cell surface expression of CD59 to the same degree asPSCA but had no effect on E25. These results support the prediction thatPSCA is a glycosylated, GPI-anchored cell surface protein.

Example 3 Isolation of cDNA Encoding Murine PSCA Homologue

The human PSCA cDNA was used to search murine EST databases in order toidentify homologues for potential transgenic and knockout experiments.One EST obtained from fetal mouse and another from neonatal kidney were70% identical to the human cDNA at both the nucleotide and amino acidlevels. The homology between the mouse clones and human PSCA includedregions of divergence between human PSCA and its GPI-anchoredhomologues, indicating that these clones likely represented the mousehomologue of PSCA. Alignment of these ESTs and 5′ extension usingRACE-PCR provided the entire coding sequence (FIG. 2).

Example 4 Isolation of Human and Murine PSCA Genes

This experiment shows that PSCA is located at chromosome 8, band q24.2.

Materials and Methods

Genomic Cloning:

Lambda phage clones containing the human PSCA gene were obtained byscreening a human genomic library (Stratagene) with a human PSCA cDNAprobe (Sambrook et al., 1989, Molecular Cloning (Cold Spring Harbor)).BAC (bacterial artificial chromosome) clones containing the murine PSCAgene were obtained by screening a murine BAC library (Genome Systems,Inc., St. Louis, Mo.) with a murine PSCA cDNA probe. A 14 kb human Not Ifragment and a 10 kb murine Eco RI fragment were subcloned intopBluescript (Stratagene), sequenced, and restriction mapped.

Chromosome Mapping by Fluorescence In Situ Hybridization:

Fluorescence in situ chromosomal analysis (FISH) was performed aspreviously described using overlapping human lambda phage clones (Rowleyet al., 1990, PNAS USA 87: 9358-9362, H. Shizuya, PNAS USA, 89:8794).

Results

Structure of PSCA Gene:

Human and murine genomic clones of approximately 14 kb and 10 kb,respectively, were obtained and restriction mapped. A schematicrepresentation of the gene structures of human and murine PSCA andLy-6/Thy-1 is shown in FIG. 8A-C. Both the human and murine genomicclones contain three exons encoding the translated and 3′ untranslatedregions of the PSCA gene. A fourth exon encoding a 5′ untranslatedregion is presumed to exist based on PSCA's homology to other members ofthe Ly-6 and Thy-1 gene families (FIG. 8A).

Human PSCA Gene Maps to Chromosome 8q24.2:

Southern blot analysis of LAPC-4 genomic DNA revealed that PSCA isencoded by a single copy gene. Other Ly-6 gene family members containfour exons, including a first exon encoding a 5′ untranslated region andthree additional exons encoding the translated and 3′ untranslatedregions. Genomic clones of human and murine PSCA containing all but thepresumed 5′ first exon were obtained by screening lambda phagelibraries. Mouse and human PSCA clones had a similar genomicorganization. The human clone was used to localize PSCA by fluorescencein situ hybridization analysis. Cohybridization of overlapping humanPSCA lambda phage clones resulted in specific labeling only ofchromosome 8 (FIG. 13). Ninety seven percent of detected signalslocalized to chromosome 8q24, of which 87% were specific for chromosome8q24.2. These results show that PSCA is located at chromosome 8, bandq24.2.

Example 5 Generation of Monoclonal Antibodies Recognizing DifferentEpitopes of PSCA Materials and Methods

Generation and Production of Monoclonal Antibodies:

BALB/c mice were immunized three times with a purified PSCA-glutathioneS-transferase (GST) fusion protein containing PSCA amino acids 22-99(FIG. 1B). Briefly, the PSCA coding sequence corresponding to aminoacids 18 through 98 of the human PSCA amino acid sequence wasPCR-amplified using the primer pair:

(SEQ ID NO: 14) 5′ primer-GGAGAATTCATGGCACTGCCCTGCTGTGCTAC(SEQ ID NO: 15) 3′ primer-GGAGAATTCCTAATGGGCCCCGCTGGCGTT

The amplified PSCA sequence was cloned into pGEX-2T (Pharmacia), used totransform E. coli, and the fusion protein isolated.

Spleen cells were fused with HL-1 myeloma cells using standard hybridomatechnique. Hybridomas that were positive for PSCA by ELISA and FACSanalysis (see Results) were subcloned. Ascites fluid was produced inC.B. 17 scid/scid mice and monoclonal antibodies (mAbs) purified using aprotein G affinity column (Pharmacia Biotech, Piscataway, N.J.). PSCAmAb 1G8 was also produced in Cell-Pharm System 100 as recommended by themanufacturer (Unisyn Technologies, Hopkinton, Mass.).

ELISA for Hybridoma Screening:

GST or PSCA-GST were immobilized on Reacti-Bind maleicanhydride-activated polystyrene plates (Pierce, Rockford, Ill.). 50 ulof hybridoma media were added to each well and incubated for 1 hour atroom temperature. Wells were washed 3 times with 200 ul PBS containing0.1% BSA and 0.05% Tween 20 and incubated for 1 hour with 100 ulanti-mouse IgG (1:4000) labeled with alkaline phosphatase (Promega,Madison, Wis.). Plates were developed with an alkaline phosphatasesubstrate (Bio-Rad, Hercules, Calif.).

Cell Culture:

LNCaP was obtained from ATCC and stably transfected with a pCDNA II(Invitrogen) expression vector containing PSCA or vector alone (Reiter,R. et al., 1998). 293T cells transiently transfected with PSCA or vectoralone were prepared as described previously (Reiter, R. et al., 1998).LAPC-9 xenograft explants were propagated in PrEGM media (Clonetics, SanDiego, Calif.) after digestion in 1% pronase for 18 min. at roomtemperature. Before FACS analysis, LAPC-9 cells were passed though a 40um cell strainer to obtain single cell suspensions.

Immunofluorescence:

Cells were grown on glass coverslips coated with poly-L-lysine.Immunofluorescence assays were carried out on permeabilized andnonpermeabilized fixed cells. For fixation, cells were treated with 2%paraformaldehyde in PBS-CM (PBS, 100 uM CaCl₂, 1 mM MgCl₂) for 30minutes in the dark, quenched with 50 uM NH₄Cl in PBS-CM-BSA (PBS, 100uM CaCl₂, 1 mM MgCl₂, 0.2% BSA) for 10 minutes, and washed twice withPBS-CM-BSA. For permeabilization, cells were treated additionally withPBS-CM-BSA-Saponin (0.075% saponin (Sigma) in PBS-CM-BSA) for 15 minutesat room temperature. Primary mAb at 2-5 mg/ml in PBS-CM-BSA (plussaponin in cases of permeabilization) was added for 60 minutes andwashed twice with PBS-CM-BSA. FITC-conjugated goat antimouse IgGantibody (1:500 diluted in PBS-CM-BSA+/−saponin; Southern Biotechnology,Birmingham, Ala.) was added for 30 minutes and washed 3 times withPBS-CM. Slides were mounted in vectashield (Vector Laboratory, Inc.,Burlingame, Calif.).

Flow Cytometry:

Cells (1×10⁶) were incubated for 30 minutes at 4° C. with 100 ul mAb at2 ug/ml in PBS containing 2% fetal bovine serum or hybridoma conditionedmedium. After washing, cells were stained with a 1:500 dilution ofFITC-conjugated goat antimouse IgG (Southern Biotechnology, Birmingham,Ala.). Data was acquired on a FACScan (Becton Dickinson) and analyzed byusing LYSIS II software.

Immunoblotting and Immunoprecipitation:

Immunoprecipitation was performed as described (Harlow, E. and Lane, D.,1988). Briefly, cells were labeled with 500 uCi of trans35 label (ICN)for 6 hours. Cell lysates were incubated with 3 ug mAb and 20 ul ofprotein A-Sepharose CL-4B (Pharmacia Biotech) for 2 hours. Forimmunoblotting, protein extracts were prepared by lysing cells in 1×SDSLaemmli sample buffer and boiling for 5 min. Proteins were separated on12.5% SDS polyacrylamide gels and transferred to nitrocellulosemembranes, washed and incubated with 2 ug mAb in 10 ml blocking buffer(5% nonfat milk in TBST). Blots were developed using the Amershamenhanced chemiluminescence detection system (Amersham, ArlingtonHeights, Ill.).

Immunohistochemistry:

Normal formalin-fixed, paraffin-embedded tissue samples were obtainedfrom the Departments of Pathology at Beth-Israel Deaconess MedicalCenter-Harvard Medical School and UCLA. Primary radical prostatectomyspecimens were selected from a previously described database(Magi-Galluzzi, C. et al., 1997). Bone metastases and matched primarybiopsy specimens were obtained from the UCLA Department of Pathology.Normal tissues were stained and scored independently at two institutionsin order to ensure reproducibility. Specimens obtained from UCLA werestained using modifications of an immunoperoxidase technique previouslydescribed (Said, J. W. et al., 1998). Antigen retrieval was performed onparaffin sections using a commercial steamer and 0.01M citrate buffer pH6.0. After incubation with PSCA mAbs for 50 min. (see below), slideswere treated sequentially with rabbit anti-mouse IgG, swine anti-rabbitIgG and rabbit anti-swine IgG, all biotin conjugated. Slides were thenincubated with strepavidin-peroxidase and antibody localizationperformed using the diaminobenzidene reaction. Specimens obtained fromBeth-Israel-Deaconess-Harvard Medical School were stained as previouslydescribed using an automated Ventana NexES instrument (Ventana MedicalSystems, Tucson, Ariz.) (Magi-Galluzzi, C. et al., 1997). Antigenretrieval was done by microwave for 15 min. in EDTA, pH 8.05 at 750W.mAbs purified at a concentration of ˜1 ug/ul from SCID ascites were usedat the following concentrations: 1G8=1:20; 3E6=1:30; 2H9=1:50;4A10=1:100; 3C5=1: 100. mAb 1G8 was produced in CellPharm System 100 andused at a concentration of 1:10. Positive controls included LAPC-9 andLNCaP-PSCA and negative controls were LNCaP and isotype-matchedirrelevant antibody. Primary biopsy specimens were available for threepatients with bone metastases. To approximate conditions ofdecalcification, slides from these specimens were treated for 20 min. inDecal-Stat (Lengers, NY) prior to staining with PSCA mAbs.

Monoclonal antibodies (mAbs) were raised against a PSCA-GST fusionprotein lacking both the amino and carboxyl terminal signal sequences ofPSCA. Positive fusions were selected by ELISA using the PSCA-GST fusionprotein and GST alone. Out of 400 hybridomas screened, 28 recognized thePSCA-GST fusion but not GST alone. These fusions were screenedsecondarily by flow cytometry of nonpermeabilized 293T cells transfectedwith PSCA and mock transfected 293T cells. Secondary screening by FACSwas done in order to select clones capable of recognizing PSCA on thecell surface, hypothesizing that these might later become useful for invivo targeting applications. Seven positive fusions were identified inthis manner (mAbs 2A2, 3G3, 4A10, 1G8, 3E6, 3C5 and 2H9), of which five(mAbs 4A10, 1G8, 3E6, 3C5 and 2H9) were subcloned and purified.

The mAbs were tested for their ability to immunoprecipitate PSCA and/orto recognize PSCA on immunoblots. All mAbs were able toimmunoprecipitate PSCA from 293T-PSCA cells, as well as from LAPC-9prostate cancer xenograft tumors that express high levels of endogenousPSCA (FIG. 37). Likewise, all mAbs detected PSCA by immunoblotting,although mAbs 2H9 and 3E6 recognized only the ˜12 kd deglycosylated formof PSCA (FIG. 34).

The location on PSCA of the epitopes recognized by the five mAbs wasdetermined by immunoblot analysis using three truncated PSCA-GST fusionsproteins. mAbs 4A10, 2H9 and 3C5 recognize an epitope residing withinthe amino-terminal portion of PSCA (i.e., amino acids 21-50); mAb 1G8recognizes an epitope within the middle region of PSCA (i.e., aminoacids 46-85); and mAb 3E6 reacts within the carboxyl-terminal portion ofPSCA (amino acids 85-99) (FIG. 15). All five mAbs are IgG as describedin FIG. 15. These results demonstrate that the five mAbs can detect PSCAin multiple assays and recognize at least three distinct epitopes onPSCA.

PSCA mAbs Stain the Cell Surface of Prostate Cancer Cells

The utility of mAbs for studying PSCA biology and for potential clinicalapplications such as in vivo targeting applications is dependent ontheir ability to recognize the antigen of interest on the plasmamembrane (Liu, H. et al., 1997; McLaughlin, P. et al., 1998; Wu, Y. etal., 1995; Tokuda, Y. et al., 1996). In order to determine the abilityof mAbs 2H9, 3E6, 1G8, 4A10 and C5 to recognize PSCA specifically on thecell surface of prostate cancer cells, LNCaP cells transfected with PSCA(LNCaP-PSCA) and LAPC-9 cells were examined by flow cytometry andindirect immunofluorescence. As with 293T-PSCA cells, all five mAbs wereable to detect PSCA on the cell surface of nonpermeabilized LNCaP-PSCAand/or LAPC-9 cells by flow cytometry (FIG. 33). Mock-transfected LNCaPand LNCaP transfected with a neomycin-alone containing vector(LNCaP-neo), neither of which expresses detectable PSCA mRNA, were bothnegative.

Immunofluorescent analysis was performed on both permeabilized andnonpermeabilized cells in order to ascertain whether PSCA proteinlocalizes to the cell surface (Liu, H. et al., 1997). NonpermeabilizedLNCaP-PSCA showed clear cell surface reactivity with mAbs 1G8, 3E6, 4A10and 3C5, but did not stain with mAb 2H9 (mAb 2H9 also did not detectPSCA on LNCaP-PSCA cells by FACS). LAPC-9 cells showed cell surfacereactivity with all five mAbs (FIG. 35). LNCaP-neo, as predicted, wasnegative both with and without permeabilization. Permeabilization ofLNCaP-PSCA and LAPC-9 resulted in both membrane and cytoplasmicstaining. All mAbs produced a punctate staining pattern on the cellsurface, which was most pronounced with mAbs 3E6, 3C5 and 4A10 (FIG.35). This pattern may reflect aggregation or clustering of PSCA toregions of the cell surface. These results demonstrate that all fivemAbs react with PSCA on the cell surface of intact prostate cancercells.

Immunohistochemical Staining of PSCA in Normal Prostate

PSCA mRNA localizes to a subset of basal cells in normal prostate,suggesting that PSCA may be a cell surface marker for prostatestem/progenitor cells (Reiter, R. et al., 1998). In order to test thepossibility that PSCA protein may be a marker of basal cells, PSCAexpression was examined immunohistochemically in paraffin-embeddedsections of normal prostate. mAbs 1G8 and 2H9 stained the cytoplasm ofboth basal and secretory cells, while mAb 3E6 reacted predominantly withbasal cells (FIG. 38). Atrophic glands, which express basal cellcytokeratins, stained strongly with all three mAbs (FIG. 38) (O'Malley,F. P. et al., 1990). mAbs 3C5 and 4A10 gave strong background stainingand/or nonspecific nuclear staining in paraffin sections and were notused further. These results suggest that although PSCA mRNA is detectedspecifically in basal cells, PSCA protein can be detected in bothepithelial cell layers (i.e. basal and secretory) of the prostate,although there are some differences in the staining patterns ofindividual antibodies.

Immunohistochemical Analysis of Normal Tissues

Our initial studies indicated that PSCA expression in men was largelyprostate-specific, with low levels of detectable RNA in kidney and smallintestine. PSCA mRNA was also detected in placenta. Theprostate-specificity of PSCA protein expression was tested byimmunohistochemical staining of 20 tissues using mAb 1G8 (see Table 1).Positive tissue staining with mAb 1G8 was confirmed with mAbs 2H9 and/or3E6 in order to ensure reproducibility with mAbs directed againstdistinct epitopes. Staining was also performed and scored independentlyat two institutions in order to confirm the results. As predicted by theRNA analysis, placenta was positive with all mAbs tested, withcytoplasmic staining detected in the trophoblasts (FIG. 39A). In kidneystaining was detected in the collecting ducts and distal convolutedtubules, but not in glomeruli (FIG. 39A). Transitional epithelium of thebladder and ureter, which had not been examined previously at the mRNAlevel, was positive with all mAbs tested (FIG. 39A). The only othertissue with significant immunoreactivity was colon, in which singlecells deep within the crypts stained intensely positive (FIG. 39A).Double staining with chromogranin indicated that these cells are ofneuroendocrine origin.

In order to confirm that mAb reactivity in bladder represented PSCA,Northern blot analysis was performed on three normal bladder samplesobtained at radical cystectomy and compared with PSCA expression inprostate, kidney and the LAPC-9 xenograft (FIG. 39B). PSCA mRNA wasdetected in bladder at levels lower than those seen in prostate,confirming the immunohistochemical result. No signal was detected in thethree kidney specimens, consistent with our previous observation thatPSCA expression in kidney is significantly lower than prostate (Reiter,R. et al., 1998). LAPC-9, a prostate cancer xenograft established from abone metastasis, expresses very high levels of PSCA mRNA compared withnormal bladder and prostate (Whang, Y. E. et al., 1998). These resultsconfirm that PSCA expression in men is largely prostate-predominant;however, there is also detectable PSCA protein expression in urothelium,renal collecting ducts and colonic neuroendocrine cells.

PSCA Protein is Expressed by a Majority of Localized Prostate Cancers

In our previous study, mRNA was expressed in ˜80% of tumors and appearedto be expressed more highly in normal than malignant glands (Reiter, R.et al., 1998). In order to determine if PSCA protein can be detected inprostate cancers and if PSCA protein levels are increased in malignantcompared with benign glands, paraffin-embedded pathological specimens ofprimary and metastatic prostate cancers were immunostained with mAb 1G8(FIGS. 21 and 28). Isolated cases were also stained with mAbs 3E6 or 2H9in order to confirm the specificity of the staining. Twelve of 15primary cancers stained positive (FIG. 21), including 2/2 casescontaining foci of high grade prostatic intraepithelial neoplasia.Staining intensity varied, with 7 cases showing equivalent staining incancer and adjacent normal glands and 5 showing significantly strongerstaining in cancer. In some cases there was strong expression in themalignant glands and undetectable staining in adjacent normal tissue(FIG. 21; patient 1). Also, there were some cases in which staining washeterogeneous, with some malignant glands staining more strongly thanothers (FIG. 21; patient 2). Overall, poorly differentiated tumorsstained more strongly than well differentiated ones, suggesting thatPSCA overexpression may correlate with increasing tumor grade (FIG. 21;patient 3). These results demonstrate that PSCA protein is expressed inprostate cancer. Consistent with our previous mRNA in situ studies, PSCAappears to be overexpressed in a significant percentage of cancers,perhaps in concert with increasing tumor grade.

This study describes the first characterization of PSCA proteinexpression using five monoclonal antibodies directed against PSCA.Because these mAbs recognize epitopes on the exterior of the cellsurface, they may have utility for prostate cancer diagnosis and therapy(Liu, H. et al., 1997). One possibility is that these mAbs could be usedto locate sites of metastatic disease, similar to the Prostascint scanwhich uses an antibody directed against PSMA (Sodee, D. B. et al.,1996). Another possibility is that they may be used to target prostatecancer cells therapeutically, either alone or conjugated to aradioisotope or other toxin. Similar approaches are currently beingevaluated using antibodies directed against extracellular epitopes onPSMA (Murphy, G. P. et al., 1998; Liu, H. et al., 1997; Liu, H. et al.,1998).

PSCA mAbs stain the cell surface in a punctate manner, suggesting thatPSCA may be localized to specific regions of the cell surface.GPI-anchored proteins are known to cluster in detergent-insolubleglycolipid-enriched microdomains (DIGS) of the cell surface (Varma, R.and Mayor, S., 1998). These microdomains, which include caveolae andsphingolipid-cholesterol rafts, are believed to play critical roles insignal transduction and molecular transport (Anderson, R. Caveolae,1993; Friedrichson, T. and Kurzchalia, T. V., 1998; Hoessli, D. C. andRobinson, P. J., 1998). Thy-1, a homologue of PSCA, has previously beenshown to transmit signals to src kinases through interaction inlipid-microdomains (Thomas, P. M. and Samuelson, L. E., 1992; Stefanova,I. et al., 1991). Preliminary subcellular fractionation experiments inour laboratory confirm the presence of PSCA in DIGS (Xavier, R. et al.,1998).

GPI-anchored proteins have also been reported to localize toprostasomes, membrane-bound storage vesicles released by prostateepithelial cells (Ronquist, G. and Brody, I., 1985). CD59, aGPI-anchored inhibitor of complement-mediated cytolysis, is found inhigh concentrations in prostasomes of normal prostate epithelial cellsand prostatic secretions (Rooney, I. et al., 1993). PSCA protein isdetected in prostate secretory cells.

Contrary to our previous finding that PSCA mRNA localized exclusively tobasal cells, the current results suggest that PSCA protein may bepresent in both basal and secretory cells. Similar differences betweenmRNA and protein localization in prostate have been described for PSMAand androgen receptor (Magi-Galuzzi, C. et al., 1997; Kawakami, M. andNakayama, J., 1997). One possible explanation for the presence of PSCAprotein in secretory cells is that PSCA mRNA is transcribed in basalprogenitor cells but that PSCA protein expression persists as basalcells differentiate into secretory cells. Another possibility is thatPSCA protein may be transferred from basal to secretory cellsposttranslationally.

Differences in staining intensity of basal and secretory cells by mAbs3E6, 1G8 and 2H9 may reflect the distinct epitopes recognized by theantibodies and/or differences in posttranslational modification of PSCAin basal and secretory cells. Supporting this possibility is theobservation that the five mAbs do not react equally with PSCA in allassays or cell lines. mAb 2H9 recognizes PSCA on the cell surface ofLAPC-9 but not LNCaP-PSCA, suggesting that the epitope recognized bythis antibody may be altered or obscured in the latter cell type. Wehave also observed that mAb 3E6 does not stain cancers as strongly asmAbs 1G8 and 2H9 in some cases, suggesting that it may react withcertain forms of PSCA preferentially.

Although largely prostate-specific in men, PSCA is also expressed atlower levels in urothelium, colonic neuroendocrine cells, and renaltubules and collecting ducts. The staining seen in renal tubules andcollecting ducts is interesting in that these structures deriveembryologically from the ureteric bud of the mesonephric duct,suggesting a possible reason for the staining patterns seen in kidney.The absence of detectable PSCA mRNA in kidney specimens may reflecteither low levels of expression or the possibility that the samples wereobtained primarily from the renal cortex, whereas the collecting ductsare located in the renal medulla.

The primary impetus for identifying prostate-specific cell surface genesis the desire to develop selective, nontoxic therapies. PSMA, another“prostate-restricted” protein, has also been shown to be expressed induodenum, colonic neuroendocrine cells and proximal renal tubules(Silver, D. A. et al., 1997). Preliminary reports of PSMA vaccinetherapy have not produced significant toxicity (Tjoa, B. A. et al.,1998).

Expression of PSCA in urothelium and kidney appears to be lower than innormal prostate and significantly less than that seen in many of theprostate cancers evaluated. Therapies directed against PSCA maytherefore be relatively selective for cancer, much as Her-2/neuantibodies primarily target breast cancers that overexpress Her-2/neu(Disis, M. L. and Cheever, M. A., 1997).

Expression of PSCA in urothelium and kidney raises the possibility thatit may be expressed in transitional and renal cell carcinomas. Twobladder cancers examined do express PSCA, one at levels similar toLAPC-9, suggesting that PSCA may be overexpressed in some cases oftransitional cell carcinoma. A more complete survey of bladder cancerspecimens will be required to test this possibility.

The data herein supports our earlier observation that PSCA is expressedin a majority of prostate cancers. Likewise, PSCA protein isoverexpressed in some prostate tumors when compared to adjacent normalglands, supporting its use as a target for prostate cancer therapy. Incontrast to the mRNA in situ studies, the current results suggest thatPSCA protein expression may correlate with cancer stage and/or grade.Similar differences between RNA and protein expression have been notedfor thymosin Beta-15 (Bao, L., et al., 1996).

TABLE 1 PSCA expression in normal tissues. Staining Tissue PositiveProstate (epithelium) Bladder (transitional epithelium) Placenta(trophoblasts) Colon (neuroendocrine cells) Kidney (tubules andcollecting duct)* Negative Kidney (glomeruli) Prostate (stroma) Bladder(smooth muscle) Testis Endometrium Small intestine Liver Pancreas BreastGallbladder Skeletal muscle Brain Peripheral nerve Bone marrow ThymusSpleen Lung Bronchus Heart *mAb 3E6 reacts with the distal convolutedtubule, while mAb 1G8 reacts with distal tubules and, in some cases,proximal tubules. ** subsequent experimental analysis also showed PSCAexpression in normal stomach tissue.

Example 6 PSCA Expression in Prostate Cancer Bone Metastases

This experiment shows that PSCA expression is amplified in bonemetastases of prostate cancer.

Materials and Methods

Horse Serum (NHS) (GIBCO #26050-070) was diluted (1/20 dilution) in 1%Casein, PBST. The antibodies of the invention that recognize PSCA werediluted in 1/100 NHS, PB ST.

The detection system included HRP-rabbit anti-mouse Ig (DAKO P260),HRP-swine anti-rabbit Ig (DAKO P217), HRP-rabbit anti-swine Ig (DAKO P164). Each were diluted 1/100 in 1/100 NHS, PBST.

3,3′-diaminobenzidine tetrahyrochloride (DAB) (Fluka) stock was made bydissolving 5 gm in 135 ml of 0.05 M Tris, pH 7.4. DAB was aliquoted into1 ml/vial and frozen at −20° C. A working solution of DAB was made byadding 1 ml of DAB to 40 ml of DAB buffer and 40 microliters of 50%H₂O₂.

DAB buffer was prepared by combining 1.36 gm Imidazole (Sigma #1-0125)with 100 ml D²-H₂O, then adjusting the pH to 7.5 with 5 N HCl. After thepH adjustment 20 ml of 0.5 M Tris pH 7.4 and 80 ml of D²-H₂O were added.

A section of a tissue/tumor known and previously demonstrated to bepositive for the antibody was run with the patient slide. This slideserved as a “positive control” for that antibody. A section of thepatient's test specimen was incubated with a negative control antibodyin place of the primary antibody. This slide served as a “negativecontrol” for the test.

The staining procedure was as follows. Bone samples were applied toslides. The slides were then baked overnight at 60° C. Slides weredeparaffinize in 4 changes of xylene for 5 minutes each and passedthrough a graded series of ethyl alcohol (100%×4, 95%×2) to tapwaterthen transferred to NBF, and fixed for 30 minutes. The fixed slides wereplaced in running tapwater for 15 minutes, transferred to 3% H₂O₂-MeOH,incubated for 10 minutes, and washed in running tapwater for 5 minutes,then rinse in deionized water.

Slides were then subjected to 0.01 M citrate buffer pH 6.0, heated at45° C. for 25 minutes, cooled for 15 min and then washed in PBS. Theslides were then rinsed in PBS and placed onto programmed DAKOAutostainer, using the following four step program. The four stepprogram is as follows. The slide is rinsed in PBS and blocked with 1/20NHS in 1% Casein in PBST for 10 minutes. Primary antibody is thenapplied and incubated for 30 minutes followed by a buffer rinse.HRP-Rabbit anti-Mouse Ig is then applied and incubated for 15 minutesfollowed by another buffer rinse. HRP-Swine anti-Rabbit Ig is appliedand incubated for 15 minutes followed by a buffer rinse. HRP-Rabbitanti-Swine Ig is applied and incubated for 15 minutes followed by abuffer rinse. DAB is then applied to the slide and incubated for 5minutes followed by a buffer rinse. A second DAB is applied andincubated for 5 minutes followed by a buffer rinse.

The slides are removed from the Autostainer and placed into slideholders, rinsed in tapwater and counter stained with Harris hematoxylin(15 seconds). The slide is then washed in tapwater, dipped inacid-alcohol, washed in tapwater, dipped in sodium bicarbonate solution,and washed in tapwater. The slides are then dehydrated in graded ethylalcohols (95%×2, 100%×3) and Propar×3 and coverslipped with Permount.

PSCA Protein is Expressed Strongly in Prostate Cancers Metastatic toBone

Prostate cancer is unique among human tumors in its propensity tometastasize preferentially to bone and to induce osteoblastic responses.Nine sections of prostate cancer bone metastases were examinedimmunohistochemically (FIG. 28). All reacted intensely and uniformlywith mAb 1G8 (and/or 3E6). In two instances, micrometastases not readilydetectable on hematoxylin and eosin sections could be seen afterstaining with mAb 1G8 (FIG. 28; patient 5). Overall, staining in bonemetastases was stronger and more uniform than in the primary tumors. Inthree cases, biopsy specimens from the primary tumors were available forcomparison. All were weakly positive for PSCA when compared with thematched bone metastasis, suggesting that PSCA expression was increasedin bone. In one biopsy specimen, weak staining was present in only asmall focus of malignant glands, while the remaining tumor was negative(FIGS. 21 and 28; Patient 4). In two cases, the biopsy specimens wereobtained 10 and 15 years prior to the bone metastasis, indicative of along latency period between the development of the primary andmetastatic lesions. To rule out the possibility that the strong stainingin bone was caused by the decalcification process used to prepare bonesections, the three primary biopsy specimens were also treated withdecalcification buffer. Although this treatment increased backgroundstaining, it did not alter epithelial reactivity significantly,indicating that the strong signal in bone was unlikely to be caused bythe decalcification process. These results suggest that PSCA may beselected for or upregulated in prostate cancer metastases to bone.

FIGS. 21-23 show the bone samples of bone metastases of prostate cancerwere positive for PSCA. Nine sections of prostate cancer bone metastaseswere examined. Consistent, intense staining was seen in nine prostatecancer bone metastases and all reacted intensely and uniformly with mAb1G8 (and/or 3E6). In two instances, the pathologist could not readilyidentify the metastasis until staining with 1G8 highlighted the lesion.Overall, staining in bone metastases appeared stronger and more uniformthan in the primary tumors.

These results suggest that PSCA may be greatly overexpressed in prostatecancer metastases to bone. This is particularly interesting since Sca-2,a close homologue of PSCA, was recently reported to suppress osteoclastactivity in bone marrow. If PSCA had similar activity, it might provideone explanation for the tendency of prostate cancer metastases toproduce an osteoblastic response, since inhibition of osteoclastactivity would tilt the balance of activity in bone to bone formation.Another possibility is that PSCA might be involved in adhesion to bone,since other Ly-6/Thy-1 family members are involved in similar processes.There was heterogeneous expression of PSCA in a number of primaryprostate cancers. These results further support the use of PSCA as anovel target for advanced disease.

One of the most intriguing results of the present study was theconsistent, intense staining seen in the nine prostate cancer bonemetastases. LAPC-9, a xenograft established from a bony metastasis, alsostained intensely for PSCA. In three patients, matched primary biopsyspecimens showed low levels of PSCA expression compared to the bonymetastases. Areas of strong PSCA expression in the primary tumors of thethree patients examined may have been missed since only biopsies wereavailable for analysis. Heterogeneous expression of PSCA was detected inat least one matched primary tumor, as well as in number of primarytumors for whom matched metastatic lesions were not available. Also, intwo cases the primary tumor was sampled at least a decade prior to thebone metastasis, raising the possibility that clones expressing highlevels of PSCA within the primary could have developed subsequent to theinitial biopsy. These results clearly demonstrate PSCA expression inbone metastases, further supporting it as a novel target for advanceddisease.

Example 7 PSCA Overexpression in Bladder and Pancreatic Carcinomas

This experiment shows that PSCA expression is higher in bladdercarcinomas than normal bladder.

Tissues from prostate, bladder, kidney, testes, and small intestine(including prostate cancer and bladder and kidney carcinomas) wereobtained from patients. These tissues were then examined for binding toPSCA using northern and western blot analyses as follows.

For northern blot analyses, tissue samples were excised and a less than0.5×0.5 cm tissue sample was quick frozen in liquid nitrogen. Thesamples were homogenized in 7 mls of Ultraspec (Biotecx, Houston, Tex.),using a polytron homogenizer using the protocol provided by Biotecx(Ultraspec™ RNA Isolation System, Biotecx Bulletin No:27, 1992).

After quantification, 20 μg of purified RNA from each sample were loadedonto a 1% agarose formaldehyde gel. Running and blotting conditions werethe same as was used in Example 1. The filters were separately probedwith labeled PSCA and an internal control, actin. Filters were washedand exposed for several hours-overnight.

For western blot analyses, tissue samples were excised and a less than0.5×0.5 cm tissue sample was taken and quickly minced and vortexed inequal volume of hot 2× Sample Buffer (5% SDS, 20% glycerol). Sampleswere incubated at 100° for 5 mins, vortexed and clarified for 30 min.Protein concentrations were determined by Biorad's DC Protein Assay kit(Richmond, Calif.). 40 μg/sample was loaded on a 12% polyacrylamideprotein gel. Transfer to a nitrocellulose filter was done by standardmethods (Towbin et al. PNAS 76:4350 (1979). A western blot was performedby incubating the filter with IG8 primary antibody followed by asecondary antibody, i.e., a goat αmouse IgG HRP. Detection was byAmersham ECL Detection kit (Arlington Heights, Ill.).

1G8 recognized and bound the PSCA on the cells surface of LAPC9 and abladder carcinoma (designated bladder (Rob)) in a western blot analysis(FIG. 6). In FIG. 6, all tissues except LAPC9 were normal. A northernblot analysis confirmed elevated PSCA in the bladder carcinoma tissue(designated bladder (Rob) (also referred to as Rob's Kid CA) and LAPC9)(FIG. 25).

A Northern blot analysis was performed, testing transcripts isolatedfrom pancreatic cancer cell lines: PANC-1 (epithelioid, ATCC No.CRL-1469), BxPC-3 (adenocarcinoma, ATCC No. CRL-1687), HPAC (epithelialadenocarcinoma, ATCC No. CRL-2119), and Capan-1 (adenocarcinoma, livermetastasis, ATCC No. HTB-79). The Northern blot was probed with a fulllength cDNA clone of PSCA which detected PSCA transcripts in twopancreatic cancer cell lines, HPAC and Capan-1 (FIG. 63).

A Western blot analysis using the PSCA mAb 1G8 detected high levels ofPSCA protein in the HPAC cell line and lower levels in Capan-1 andASPC-1 (adenocarcinoma, ascites, ATCC No. CRL-1682) (FIG. 64).

Example 8 PSCA Gene Amplification in Prostate Cancer

This experiment shows that PSCA gene copy number is increased similar toan increase in copy number of c-myc (FIG. 17). This is important becausec-myc amplification correlates with poor outcome. Thus, the datasuggests that PSCA amplification may also be a predictor for pooroutcome.

FISH with Chromosome Enumeration Probes and a Probe for c-Myc.

The method of FISH is well known (Qian, J. et al., “ChromosomalAnomalies in Prostatic Intraepithelial Neoplasia and Carcinoma Detectedby Fluorescence in vivo Hybridization,” Cancer Research, 199555:5408-5414.) Briefly, tissue sections (samples 34 and 75 were from twopatients) were deparaffinized, dehydrated, incubated in 2×SSC at 75° C.for 15 min, digested in pepsin solution [4 mg/ml in 0.9% NaCl (pH 1.5)]for 15 min at 37° C., rinsed in 2×SSC at room temperature for 5 min, andair-dried.

Directly labeled fluorescent DNA probes for PSCA and for the 8q24(c-myc) region were chosen. The PSCA cDNA (FIG. 1A) was used to identifya 130 kb bacterial artificial chromosome (bac) clone (PSCA probe) thatin turn was used in the FISH analysis in accordance with themanufacturer's protocol (Genome Systems Inc.) The bac clone soidentified and used in the FISH analysis was BACH-265B12 (GenomeSystems, Inc. control number 17424).

Dual-probe hybridization was performed on the serial 5-μm sections usinga SG-labeled PSCA probe together with a SO-labeled probe for 8q24(c-myc). Probes and target DNA were denatured simultaneously in an 80°C. oven for 5 min. and each slide was incubated at 37° C. overnight.

Posthybridization washes were performed in 1.5 M urea/0.1×SSC at 45° C.for 30 min and in 2×SSC at room temperature for 2 min. Nuclei werecounter-stained with 4.6-diamidino-2-phenylindole and anilfade compoundp-phenylenediamine.

The number of FISH signals was counted with a Zeiss Axioplan microscopeequipped with a triple-pass filter (102-104-1010; VYSIS). The number ofc-myc signals and PSCA signals were counted for each nucleus, and anoverall mean c-myc:PSCA ratio was calculated. Results are shown in FIG.17.

The results show that PSCA gene copy number increased in prostate cancersamples (FIG. 17). The PSCA gene is located at 8q24.2. The increase ingene copy number is due to both a gain in chromosome 8, andamplification of the PSCA gene (FIG. 17). Interestingly, the increase inPSCA gene copy number is similar to an increase in gene copy number ofc-myc (FIG. 17) which is also located at 8q24. A previous study hasdemonstrated that a gain of chromosome 8 and amplification of c-myc arepotential markers of prostate carcinoma progression (R B Jenkins et at1997 Cancer Research 57: 524-531).

Example 9 Reporter Gene Construct Using the hPSCA 9 kb Upstream Regionto Drive Luciferase Expression

The 14 kb Not I genomic fragment encoding the human PSCA gene wasisolated from λFIXII library encoding human genomic DNA (Stratagene), byscreening the library with a full length human PSCA cDNA probe, asdescribed in example 4 (Sambrook et al., 1989, Molecular Cloning (ColdSpring Harbor). The 14 kb human PSCA genomic fragment includes 9 kb ofPSCA upstream sequences that was used to drive expression of a reportergene.

The reporter gene vectors are depicted in FIG. 42 and were constructedas follows. The 14 kb Not I fragment was sub-cloned from the λ vectorinto a Bluescript KS vector (Stratagene), resulting in the pBSKS-PSCA(14 kb) construct. The PSCA upstream sequence was subcloned frompBSKS-PSCA (14 kb) by PCR amplification using a primer corresponding tothe T7 sequence contained within the Bluescript vector, and a primercorresponding to a sequence contained within PSCA exon 1 (primerH3hPSCA3′-5, the sequence of this primer is as follows: The sequence ofH3hPSCA3′-5 is 5′-gggaagcttgcacagccttcagggtc-3′ (SEQ ID NO:18). Theprimer corresponding to PSCA exon 1 contained an introduced HindIIIsequence to allow further subcloning following PCR amplification. Theresulting amplified fragment was digested with HindIII and was subclonedinto the pGL3-basic vector (Promega) to generate pGL3-PSCA (7 kb) whichwas used to generate a series of deletion reporter gene constructscontaining varying lengths of PSCA upstream sequences operatively linkedto the luciferase gene (FIG. 42). The deleted portions of the PSCAupstream regions were obtained by subcloning restriction fragments frompGL3-PSCA (7 kb). The PSCA upstream region between −9 kb and −7 kb wassubcloned from the pBSKS-PSCA (14 kb) construct, the Not I site wasconverted into a blunt end by Klenow and the fragment was cloned intothe SacI/HindIII sites of pGL-PSCA (7 kb) in order to obtain thepGL3-PSCA (9 kb) construct. The reference to the sequences upstream ofthe PSCA coding region, such as −9 kb and −6 kb (etc.), are relative tothe ATG start translation codon. The reporter gene constructs pGL3-PSCA(9 kb), pGL3-PSCA (6 kb), pGL3-PSCA (3 kb), and pGL3-PSCA (1 kb) wereoperatively linked to the luciferase gene (FIG. 42). Plasmid, pGL3-CMVcontains the cytomegalovirus promoter (Boshart, M. et al., 1985 Cell41:521-530) linked to the luciferase gene and was used as a positivecontrol. Also, plasmid pGL3 contains no promoter sequence and was usedas a negative control plasmid.

Example 10 Transfection Assay Using a Reporter Gene Construct Containingthe hPSCA Upstream Region

Triplicate dishes of prostate and non-prostate cell lines weretransfected by Tfx50 (Boeringer Manheim) with the PSCA constructpGL3-PSCA (9 kb), or the positive control construct, pGL3-CMV bothdescribed in Example 9 above, and assayed for luciferase activity (FIG.43). The cells and cell lines transfected include PrEC(androgen-independent prostate basal cell), LNCaP (androgen-dependentprostate secretory cell line), LAPC4 (androgen-dependent prostate cellline), HT1376 (bladder cell line), and 293T (kidney cell line).Expression activities of the constructs are expressed as a percentage ofthe activity of the CMV promoter. Standard errors are indicated abovethe bars.

The results show that 9 kb of human PSCA upstream sequences drivesexpression of the luciferase gene in a tissue-specific manner similar tothe mRNA expression patterns seen for native hPSCA shown in FIG. 10(Example 1). Luciferase was readily detectable in bothandrogen-dependent and androgen-independent prostate cell lines andbladder. Luciferase was also detectable, although at a lower level, inkidney cells.

Example 11 Identification of Regulatory Elements within the PSCAUpstream Region

Triplicate dishes of PrEC (Clonetech) or LNCaP cells were transfectedwith the reporter gene constructs or the positive control constructdescribed in Example 9 above, and assayed for luciferase activity. Thereporter gene constructs comprise various lengths of the hPSCA upstreamregion operatively linked to the luciferase gene. The positive controlconstruct, pGL3-CMV, comprises the CMV promoter operatively linked tothe luciferase. The cells were transfected using a Tfx50 transfectionsystem (Promega). Expression of luciferase in the transfected cells wereassayed using a Dual Luciferase Reporter Assay System (Promega), and thelevel of luciferase expression was measure a relative luciferase unit(RLU).

The ability of the various lengths of the hPSCA upstream region to driveluciferase expression are expressed as a percentage of the activity ofthe positive control construct containing the CMV promoter. Standarderrors are indicated.

The results shown in FIG. 44 demonstrate that 3 kb of hPSCA upstreamsequences drives expression of luciferase in both PrEc and LNCaP cells,but the level of detectable luciferase is 6 times higher in the LNCaPcells compared to the PrEC cells. This comparison was based on the levelof detectable luciferase. In contrast, 1 kb of hPCSA upstream sequencesdid not drive expression of luciferase in either cell line.

Example 12 A Targeting Vector

A targeting vector was designed to delete the endogenous PSCA codingregion, by homologous recombination. FIG. 40 depicts a targeting vectorfor the mouse PSCA gene, and the strategy for using the targeting vectorto delete the endogenous PSCA gene contained in a mouse cell. Atargeting vector comprising a 12 kb SpeI fragment containing mouse PSCAupstream sequences, a NotI/EcoRI fragment containing the PGK promoteroperatively linked to a neo^(r) gene from the pGT-N29 vector (NewEngland BioLabs), and a 3.5 kb BstXI/XhoI fragment containing mouse PSCAdownstream sequences. Constitutive expression of the neomycin resistancegene is controlled by the PGK promoter, and allows antibiotic selectionof the targeted cells that contain the targeting vector.

As understood by one skilled in the art, the targeting vector describedhere includes but is not limited to the neo^(r) gene for selection ofthe cells that contain the targeting vector or can contain no selectablereporter gene. The targeting vector can also be used to generatetransgenic mice, known in the art as knock-in or knock-out mice,depending on whether the targeting vector contains a reporter gene ornot, respectively. The transgenic mice can be used as an animal model tostudy the function of the PSCA gene in prostate development of mice.

As an example that is not intended to be limiting, the targeting vectorwas used to delete the wild type endogenous genomic mouse PSCA codingsequences in embryonic stem cells (ES) cells to generate cells that areheterozygous, containing a deleted PSCA gene. For example, theheterozygous cells generated using the targeting vector arePSCA+/neo^(r) as shown by the results in FIG. 40. The phenotype of theheterozygous cells or transgenic mice can be compared with that of wildtype PSCA cells or animals.

The targeting vector was constructed as follows. The ends of the 12 kbSpeI fragment containing the PSCA upstream and part of exon 1 sequenceswas blunt-ended and linked to the blunt-ended NotI/EcoRI fragment frompGT-N29 (BioLabs) containing the neomycin-resistance gene. The 3′ end ofthe neomycin-resistance gene was linked to a blunt-ended 3.5 kbBstXI/XhoI fragment containing part of PSCA exon 3 and the downstreamsequences. The resulting fragment was cloned into pGT-N29 to generatethe targeting vector pGT-N29-mPSCA5′/3′.

The targeting vector was transfected into ES cells by electroporationusing the method described in the following: Teratocarcinomas andEmbryonic Stem Cells; A Practical Approach. IRL Press, Oxford (1987).Neomycin-resistant cells were selected and genomic DNA was isolated fromthe selected cells. A genomic Southern analysis was performed todetermine the outcome of the homologous recombination reaction. 10 μg ofDNA from the homologous recombination reaction and non-targeted ES cellswere digested with EcoRI and analyzed by the Southern blot method(Southern, E M 1975 J. Molec. Biol. 98:503). The blot was probed with aXhoI/EcoRI fragment that contains sequences 3′ to the PSCA codingregion. The results show that the probe detects a 10 kb fragment thatcorresponds to the control non-targeted cells that are PSCA+/PSCA+, anda 4 kb fragment that corresponds to the targeted cells that areheterozygous and contain PSCA+/neo^(r).

Example 13 Transgenic Mouse Models for Prostate Cancer

The present invention contemplates a strategy to generate transgenicmouse models for prostate cancer, using the upstream regions of the PSCAgene to drive expression of an oncogene, to induce tumor formation inprostate basal cells. As shown in FIG. 41, the strategy involvesadministration, e.g., microinjection, of a chimeric oncogene vector,comprising the upstream region of the PSCA gene operatively linked to atransgene that encodes a gene product that induces formation of a tumor.Other researchers have used this technique, using different prostate andnon-prostate regulatory sequences operatively linked to an oncogene. Forexample, C3(1) is a prostate-predominant regulatory sequence (Moroulakouet at 1994 Proc. Nat. Acad. Sci. 91: 11236-11240) and probasin is aprostate-specific regulatory sequence (Greenberg et al 1995 Proc. Nat.Acad. Sci. 92: 3439-3443), and both of these regulatory sequences driveexpression of a transgene in prostate secretory cells. Cryptdin2 is asmall-intestine predominant regulatory sequence (Garagenian et at Proc.Nat. Acad. Sci. 95: 15382-15387) that caused expression of an oncogenein prostate endocrine cells. In contrast, the present inventioncontemplates using the PSCA upstream region to drive expression of anoncogene in prostate basal cells, in order to generate a transgenicmouse model for prostate cancer.

The clinical characteristics of the induced prostate tumor can beanalyzed and compared with known characteristics of tumors caused by theparticular oncogene used in constructing the chimeric oncogene vector.In addition, various tissues and organs of the transgenic mouse can beanalyzed by DNA, RNA and proteins analyses to ascertain the presence andexpression patterns of the chimeric oncogene vector.

Example 14 Transgenic Mice Carrying Chimeric Vectors Comprising hPSCAUpstream Sequences and a Transgene

The expression patterns of transgenes under the control of hPSCAupstream regions will be tested. Toward this end, chimeric mice carryingchimeric vectors comprising hPSCA upstream sequences and a transgenehave been generated. Chimeric vectors comprising 9 kb or 6 kb of hPSCAupstream sequences operatively linked to a transgene were constructed,and are schematically represented in FIG. 45. The transgenes includedgreen fluorescent protein cDNA (GFP, Clontech) linked to the SV40polyadenylation sequence (PSCA (9 kb)-GFP and PSCA (6 kb)-GFP), greenfluorescent protein cDNA linked to a 3′ region of the human growthhormone that contains an intron cassette that confers stability to mRNA(PSCA (9 kb)-GFP-3′hGH and PSCA (6 kb)-GFP-3′hGH) (Brinster et at 1988PNAS 85: 836-840), and the genomic fragment encoding SV40 small andlarge T antigen including an intron (PSCA (9 kb)-SV40TAG and PSCA (6kb)-SV40TAG) (Brinster et at 1984 Cell 37:367-379).

The chimeric vectors were used to generate a line of founder transgenicmice. Linearized chimeric vectors were microinjected into fertilizedmouse eggs derived from intercrosses of C57BL/6X C3H hybrid mice.Founder mice that carried the chimeric vector were identified bySouthern analysis of tail DNA, using GFP cDNA or SV40 genomic DNA as aprobe. The number of founders of each transgenic mouse line is indicatedon the right panel of FIG. 45.

Example 15 hPSCA Upstream Sequences Drives Expression of Transgene inTransgenic Mice

Two independent founder mice carrying PSCA (9 kb)-GFP transgene werebred to Balb/c mice to obtain their offspring. At age of 8 weeks and 12weeks, male and female transgenic or non-transgenic littermates weresacrificed. After sacrifice, all urogenital and other tissues weretested for GFP expression by observing the fixed tissues underfluorescent illumination. The results shown in FIG. 46 show greenfluorescent images of prostate, bladder and skin tissues from a nontransgenic and a transgenic mouse. One out of two founder linesexpressed GFP protein in prostate, bladder and skin (FIG. 46). Tissuesthat did not express GFP include: seminal vesicle, liver, stomach,kidney, lung, brain, testis, pancreas, heart, skeletal muscle, smallintestine, colon, placenta.

Example 16 Transcript Expression Pattern of PSCA in Human and MouseTissue

The upper panel of FIG. 47 shows a human multiple tissue Northern blot(obtained from Clonetech), probed with a full length human PSCA cDNAprobe. The results demonstrate that human PSCA transcripts are abundantin prostate, and less abundant but readily detectable in placenta, butnot detectable in spleen, thymus, testis ovary, small intestine, colon,peripheral blood leukocytes (PBL), heart, brain, lung, liver, muscle,kidney and pancreas.

The lower panel of FIG. 47 shows an ethidium bromide-stained agarose gelof RT-PCR analysis of murine PSCA transcript expression patterns invarious mouse tissues. The RT-PCR was prepared using Ultraspec.RNA(Biotex), and cDNA cycle kit (Invitrogen). Primers corresponding to aregion within exon 1 and exon 3 of PSCA were used to amplify a 320 bpfragment. The exon 1 primer sequence is as follows: 5′ primer:5′-TTCTCCTGCTGGCCACCTAC-3′ (SEQ ID NO:7). The exon 3 primer sequence isas follows: 3′ primer: 5′-GCAGCTCATCCCTTCACAAT-3′ (SEQ ID NO:8). As acontrol, to demonstrate the integrity of the RNA samples isolated fromthe various mouse tissues, a 300 bp G3PD fragment was amplified.

The results shown in the lower panel of FIG. 47 demonstrate that murinePSCA transcripts are detectable in dorsal/lateral prostate, ventralprostate, bladder, stomach (cardiac, body and pyloric), and skin. Incontrast, murine PSCA transcripts are not detectable in anteriorprostate, ventral prostate, seminal vesicle, urethra, testis, kidney,duodenum, small intestine, colon, salivary gland, spleen, thymus, bonemarrow, skeletal muscle, heart, brain, eye, lung and liver. The G3PDHresults demonstrate that the transcripts isolated from various mousetissue were intact.

Example 17 Immunohistochemical Evidence of High Level Overexpression ofPSCA in Bladder Cancer

The following example demonstrates that PSCA protein is highlyoverexpressed in various grades of bladder carcinoma as determined byimmunohistochemical staining of paraffin-embedded bladder and bladdercarcinoma tissue sections using PSCA mAb 1G8. Specifically, thefollowing four tissues were examined: (A) normal bladder, (B)non-invasive superficial papillar, (C) carcinoma in situ (a high gradepre-cancerous lesion, (D) invasive bladder cancer.

The results are shown in FIG. 62A-D. PSCA is expressed at low levels inthe transitional epithelium of normal bladder tissue. Very high levelexpression was detected in the carcinoma in situ sample, in all celllayers. In the invasive bladder carcinoma sample, very strong stainingwas seen, again in all cells. Lower level staining was observed in thesuperficial papillar sample. These results suggest that PSCA expressionlevels may correlate with increasing grade.

In addition to the above study, preliminary results from animmunohistochemical analysis of PSCA expression in a large number ofbladder and bladder carcinoma tissue specimens indicates the following(1) normal bladder expresses low levels of PSCA in the transitionaleptihelium; similar levels of expression are seen in low grade,papillary, noninvasive lesions; (2) carcinoma in situ, a high grade,often quite aggressive precancerous lesion, is almost always (90%)intensely positive for PSCA in all cells; (3) PSCA is expressedintensely by ˜30% of invasive cancers, i.e. overexpressed when comparedto normal bladder; and (4) metastases are intensely positive for PSCA.

Example 18 PSCA Monoclonal Antibody Mediated Inhibition of ProstateTumors In Vivo

The following examples demonstrate that unconjugated PSCA monoclonalantibodies inhibit the growth of human prostate tumor xenografts grownin SCID mice, both when administered alone or in combination.

A. Tumor Inhibition Using Multiple Unconjugated PSCA mAbs—Study 1

Materials and Methods Anti-PSCA Monoclonal Antibodies:

Murine monoclonal antibodies were raised against a GST-PSCA fusionprotein comprising PSCA amino acid residues 18-98 of the PSCA amino acidsequence (FIG. 1B) and expressed in E. coli, utilizing standardmonoclonal antibody production methods. The following seven anti-PSCAmonoclonal antibodies, produced by the corresponding hybridoma celllines deposited with the American Type Culture Collection on Dec. 11,1998, were utilized in this study:

Antibody Isotype ATCC No. 1G8 IgG1 HB-12612 2H9 IgG1 HB-12614 2A2 IgG2aHB-12613 3C5 IgG2a HB-12616 3G3 IgG2a HB-12615 4A10 IgG2a HB-12617 3E6IgG3 HB-12618

Antibodies were characterized by ELISA, Western blot, FACS andimmunoprecipitation for their capacity to bind PSCA. FIG. 49A-B showsepitope mapping data for the above seven anti-PSCA mAbs as determined byELISA and Western analysis, as described in the accompanying figurelegend, demonstrating that the seven antibodies recognize differentepitopes on the PSCA protein. Immunohistochemical analysis of prostatecancer tissues and cells with these antibodies is described in Examples5 and 6 infra.

Antibody Formulation:

The monoclonal antibodies described above were purified from hybridomatissue culture supernatants by Protein-G Sepharose chromatography,dialyzed against PBS, and stored at −20° C. Protein determinations wereperformed by a Bradford assay (Bio-Rad, Hercules, Calif.).

A therapeutic antibody cocktail comprising a mixture of the sevenindividual monoclonal antibodies, as indicated in Table 2, below, wasprepared and used for the treatment of SCID mice receiving subcutaneousinjections of LAPC-9 prostate tumor xenografts. Mouse IgG, purchasedfrom ICN (Costa Mesa, Calif.) was used as non-specific control antibody.Prior to injection into mice, all antibodies were sterilized using a0.22-micron filter.

TABLE 2 Anti-PSCA Antibody Cocktail Amount Monoclonal Antibody Isotype(% of total) 1G8 IgG1 2.0 mg (16.7%) 2H9 IgG1 1.0 mg (8.3%) 2A2 IgG2a2.5 mg (20.8%) 3C5 IgG2a 2.0 mg (16.7%) 3G3 IgG2a 2.5 mg (20.8%) 4A10IgG2a 1.5 mg (12.5%) 3E6 IgG3 0.5 mg (4.2%)Introduction of Prostate Cancer Xenografts into SCID Mice:

The human prostate cancer xenograft line LAPC-9, which expresses veryhigh levels of PSCA, was used to produce tumors in SCID mice (PCTApplication No. WO98/16628, supra; Klein et al., 1987, supra).

For injection into IcR-SCID mice (Taconic Farms, Germantown, N.Y.), asingle-cell suspension of LAPC-9 was prepared as follows. An LAPC-9xenograft tumor of approximately 2.0 g in size was harvested from a SCIDmouse, minced into very small pieces using scissors and forceps, washedonce in RPMI, and digested in a 1% solution of pronase for 20 minutes.After digestion, the cell suspension was washed twice in RPMI, andresuspended in 10 ml of PrEGM medium (Clonetics, Walkersville, Md.).After overnight incubation, the cells were harvested and washed once inPrEGM, then passed through a 200-micron nylon filter to remove largeclumps and debris. Cells passing through the filter were collected,centrifuged, and resuspended in PrEGM medium. Cells were then counted,and the appropriate number of cells was transferred to a new tube,centrifuged, and resuspended at 2× concentration in RPMI. An equalvolume of ice cold Matrigel was then added to the cell suspension, andthe suspension was kept on ice prior to injection. For injection, maleIcR-SCID mice were shaved on their flanks, and each mouse received asingle subcutaneous (s.c.) injection of 1×10⁶ cells in a volume of 100μl on the right flank. Mice injected with tumor cells were treated witheither control antibodies or the anti-PSCA monoclonal antibodypreparation as described below.

Treatment Protocol:

Twenty SCID mice injected with tumor cells were treated with eithercontrol antibodies (mouse IgG) or the anti-PSCA monoclonal antibodycocktail (above) as follows. Ten mice were treated with mouse IgGcontrol antibody and ten mice were treated with the anti-PSCA monoclonalantibody preparation. Injections of 200 μg of the mouse IgG controlantibody or the anti-PSCA monoclonal antibody cocktail were administeredintraperitoneally on days −1, +3, +7, +11, +14, and +21 relative to theinjection of the tumor cells. Growth of LAPC-9 tumors was followed bycaliper measurements to determine tumor volumes on days +32, +35, +39,+42, +47, +54 and +61 relative to injection of tumor cells. In addition,mice were periodically bled for assaying circulating PSA levels using acommercially available PSA test (American Qualex, San Clemente, Calif.).

One of the mice in the control group (mouse #2) expired during thecourse of the study and had no detectable tumor at the time.

Results

SCID mice receiving a subcutaneous injection of the LAPC-9 prostatecancer xenograft were treated with either the anti-PSCA mAb preparationor mouse IgG control antibody, as described above. Palpable tumors firstappeared in the mouse IgG control group at 4 weeks after tumor cellinjection. Tumor volume measurements were initiated on day +32.

The results, which are tabulated in Table 3, below, as well as presentedgraphically in FIG. 48, show that all of the control mAb-treated micedeveloped tumors (9 out of 9 surviving, mouse #1, #3-10), but that noneof the anti-PSCA mAb treated mice developed any detectable tumor growth(0 out of 10, mouse #11-20). The control-treated animals developedsignificant tumors rapidly in most instances, and these mice experiencedconstant tumor growth leading to progressively larger tumor sizes withtime. By day 54, all control-treated mice had developed detectabletumors. In sharp contrast to the control-treated group, none of the tenmice treated with the anti-PSCA mAb preparation developed detectabletumors, even after 61 days post xenograft injection.

TABLE 3 Recorded tumor volume (mm³) measurements DAYS MOUSE #* 32 35 3942 47 54 61 1 416^(•)   576 578 720 810 1045 1080 2 0 0 0 0 3 100  269.5450 476 544 648 810 4 0 0 0 0 0 87.5 151.3 5 338  420 800 900 1087 12652002 6 216  250.3 504 476 612 850.5 1050 7 252  472.5 637.5 720 720 7201306 8 336  532 560 693 1080 1365 1617 9 0 160.9 225 294 478 640 900 100 0 195 294 341 504 769.5 11 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 13 0 0 0 0 00 0 14 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 17 0 0 0 0 0 0 018 0 0 0 0 0 0 0 19 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 *Mice # 1-10represent the group treated with the mouse IgG control antibody. Mice #11-20 represents the group treated with the anti-PSCA mAb cocktail.*Tumor volume corresponds to length (L) × width (W) × height (H)measurements in mm. To determine the ellipsoid volume represented inFIG. 1, which accurately represents tumor mass (Tomayko and Reynolds,1989), we used the formula L × W × H × ½.

Clinically, the control treated mice all displayed visual symptoms ofprogressively poor health as tumors developed and expanded. In contrast,the mice in the anti-PSCA mAb treatment group remained active, vigorous,and generally healthy in appearance throughout the treatment period,suggesting no apparent toxicity or obvious side-effects were associatedwith the treatment.

In addition to tumor volume, mice were bled for determination ofcirculating PSA. Circulating PSA levels correlated with increasing tumorvolumes in the control group, whereas no detectable PSA was observed inthe anti-PSCA mAb treated group throughout the experiment.

B. Tumor Inhibition Using Multiple Unconjugated PSCA mAbs—Study 2

To verify the results described in Example 18, supra, a newly preparedanti-PSCA mAb cocktail was evaluated for growth inhibition of LAPC-9tumor xenografts in vivo, essentially as described above. Briefly, a newbatch of each mAb was prepared and mixed together according to theproportions presented in Table 4. All antibodies were tested for PSCAreactivity. SCID mice received a subcutaneous injection of LAPC-9xenograft cells as described above. The mice were treated with either acocktail of anti-PSCA mAb, or control preparations of mouse IgG orpurified bovine IgG. A bovine IgG control group was included in thisstudy in order to study the effect of bovine IgG co-purified with theanti-PSCA antibodies on protein G-sepharose. Two hundred micrograms ofantibody was administered to each mouse by intraperitoneal injection ondays −1, +3, +7, +11, +14, and +21 relative to the injection of thetumor cells. Tumor volume corresponding to length (L)×(W)×(H) in mm wasmonitored by caliper measurements, and serum was collected at weeklyintervals. To determine the ellipsoid volume of the tumors, whichaccurately represents tumor mass, we used the formula L×W×H×1/2 (Tomaykoand Reynolds, 1989).

TABLE 4 Anti-PSCA antibody cocktail 2 Amount Monoclonal Antibody Isotype(% of total) 1G8 IgG1 8.0 mg (16.7%) 2H9 IgG1 4.0 mg (8.3%) 2A2 IgG2a10.0 mg (20.8%) 3C5 IgG2a 8.0 mg (16.7%) 3G3* IgG2a 10.0 mg (20.8%) 4A10IgG2a 6.0 mg (12.5%) 3E6 IgG3 2.0 mg (4.2%) *One of the monoclonalantibody preparations used to formulate this cocktail, 3G3, demonstratedweak reactivity.

The results of this study are presented in FIG. 53 and confirm theresults generated from the study described in Example 18-A, supra.Animals in the anti-PSCA treated group experienced significantinhibition of tumor cell growth compared with both of the controlgroups. No detectable difference in tumor growth was observed in micethat received either bovine IgG or murine IgG. The tumors in the controlgroups grew at equal rates and with similar latency. In contrast, LAPC-9tumors in mice receiving the anti-PSCA antibody cocktail exhibited alonger latency, a significantly slower rate of growth and smaller sizesat the end of the experiment. The average tumor volume at the final timepoint was 1,139 mm³ for mice treated with murine IgG (day 46), 1091 mm³for mice treated with bovine IgG (day 42) and 391 mm³ for anti-PSCAtreated mice (day 46). Due to the large tumor sizes in the bovine IgGtreated group, these mice were sacrificed earlier than mice in the othergroups. In addition, tumor volume correlated with PSA levels in theserum of the treated mice. Some mice receiving anti-PSCA antibodiesshowed very small tumors or no tumor growth at all, as was previouslyobserved in the study described in Example 1, supra. No apparenttoxicity was associated with administration of any of this antibodycocktail preparation, consistent with the study described in Example18-A.

C. Tumor Inhibition In Vivo Using Single Unconjugated PSCA mAbs

Materials and Methods:

Several of the monoclonal antibodies described herein were studied fortheir ability to inhibit the growth of prostate tumor xenografts intheir unconjugated (or, “naked”) form using the previously describedtumor challenge assay (see Examples 18-A and 18-B, above). Generally,the studies were conducted as described above, with slight modificationsas described in the results sections presented below for each of theantibodies assayed.

C1: PSCA mAb 1G8

Anti-PSCA monoclonal antibody 1G8 is an IgG1 isotype antibody. Theantitumor effect of 1G8 was evaluated using the LAPC-9 xenograft andmouse IgG as a control. The results presented in FIG. 54 demonstratethat treatment of mice with the 1G8 antibody inhibited tumor growth.Specifically, the average tumor volume at the final time point for thecontrol group was 854 mm³ versus an average tumor volume of 335 mm³ forthe 1G8 antibody treated group. These results show that the 1G8monoclonal antibody can inhibit the growth of prostate tumors when usedalone. As with the studies described supra, there was no apparenttoxicity associated with the treatment of these animals with the 1G8mAb.

The effect of the 1G8 monoclonal antibody on the growth of prostatecancers generated with PC-3 cells was also determined. PC-3 cells do notexpress PSCA. As shown in FIG. 65, the 1G8 antibody had no effect on thedevelopment of PC-3 xenograft tumors, in sharp contrast to its effect onPSCA-expressing LAPC-9 xenografts. These results clearly show that the1G8 antibody is inhibiting tumor cell growth through the PSCA antigen.

C2: PSCA mAbs 2A2 and 2119

Two anti-PSCA monoclonal antibodies of different isotypes were evaluatedsimultaneously for prostate tumor growth inhibition in vivo. Anti-PSCAmAbs 2A2 (IgG2a isotype) and 2H9 (IgG1 isotype) were tested for prostatetumor inhibition as described in Example 18-C1, immediately above. Theresults presented in FIG. 55 demonstrate striking inhibition of tumorcell growth in the anti-PSCA mAb treated groups versus the controlgroups. Specifically, the average tumor volume at the final time pointwas 483 mm³ for mice treated with murine IgG (day 42), 49 mm³ for micetreated with the 2A2 mAb (day 42), and 72 mm³ for the mice treated with2H9 mAb (day 42). More significantly, tumor incidence was 6/6 mice inthe mouse IgG control group, versus 2/7 for the 2A2-treated group and1/7 for the 2H9-treated group. In the 2A2 treated group, the first tumorappeared at day 25 and the second tumor at day 42. In the 2H9 treatedgroup the single tumor present appeared at day 21. In the mouse IgGcontrol group, 4/6 of the mice had developed tumors by day 21. As withthe in vivo studies described above, there was no apparent toxicityassociated with the treatment of these animals with the 2A2 or 2H9 mAbs.

PSA levels in the serum of the treated mice were significantly lowerthan in control mice, and correlated directly with tumor volume (FIG.56). At week 6, the mean PSA serum level in the mouse IgG control groupwas 35 ng/ml, 2 ng/ml in the 2A2 group, and 8 ng/ml in the 2H9 group.

This study further supports the conclusion that a single “naked”anti-PSCA monoclonal antibody is sufficient for anti-tumor activity. Inaddition, these data demonstrate that mAbs recognizing different PSCAepitopes are effective, and that the anti-tumor effect is not dependentupon a single IgG isotype since both IgG1 (1G8, 2H9) and IgG2a (2A2)mAbs inhibited tumor growth.

C3: PSCA mAbs Exert Growth Inhibitory Effect Specifically Through PSCA

In order to demonstrate that PSCA mAbs exert tumor growth inhibitionspecifically through the PSCA protein, a tumor inhibition study with the1G8 mAb and PC-3 tumor xenografts was conducted. PC-3 cells do notexpress endogenous PSCA. This study was conducted as described inSection C1 of this Example, above. The results, shown in FIG. 65, showthat the PSCA mAb 1G8 had no effect on the growth of PC-3 tumors in miceover a 40 day period. The results are shown, for comparison, togetherwith a parallel study of the effect of 1G8 on LAPC-9 prostate tumorxenografts (Example C1, above).

C4: PSCA mAb 3C5 Inhibits the Growth of Established LAPC-9 ProstateTumors in Vivo

In order to determine whether PSCA mAbs could effect growth ofestablished tumors, the following study was conducted. Briefly, a cohortof SCID mice were injected with 10⁶ LAPC-9 cells SQ, essentially asdescribed in examples C1 and C2. After tumors reached a size ofapproximately 100 cubic millimeters, mice were segregated into twogroups, a control group receiving mouse IgG and a treatment groupreceiving PSCA mAb 3C5. Each mouse was injected IP with control IgG or3C5 mAb according to the following protocol: 1 mg per injection, threetimes per week for the first 2 weeks, followed by two times per week inthe third week. Tumor volume and PSA measurements were determined asabove. The results, shown in FIG. 57, indicate that the 3C5 mAb inhibitsthe growth of established LAPC-9 prostate tumors in vivo. In at leastsome of the treatment group mice, tumor regression up to 50% of theinitial, pre-treatment size of the tumor was observed.

Example 19 In Vitro Assays for Characterizing PSCA Monoclonal Antibodies19-A: Antibody-Dependent Cell Cytotoxicity Assay

To determine if the anti-PSCA mAbs sensitize tumor cells to ADCC, thefollowing assay is performed. First, for NK cell mediated ADCC, spleencells from SCID mice are cultured for 5 days in vitro as described byHooijberg et al., 1995, Cancer Res. 55: 2627-2634. The activated cellsare then co-cultured with ⁵¹Cr-labeled LAPC-9, LNCaP-PSCA, or LNCaPtarget cells for four hours in the presence of either anti-PSCA mAbs ora control mouse IgG. LNCaP serves as a negative control in all assayssince it does not express PSCA. If single mAbs are used, the respectivemouse IgG isotype control is also used. NK activity of the activatedspleen cells is determined by incubation with the murine NK-sensitivetarget YAC-1. In all cases, killing is determined by ⁵¹Cr-release intothe medium. Spontaneous release is determined after incubation oflabeled cells only, and total release by incubation of labeled cellswith 5% Triton X-100. The percent of specific cell lysis is determinedby:

${\% \mspace{14mu} {Cell}\mspace{14mu} {Lysis}} = \frac{{{{Experimental}\mspace{14mu}}^{51}{Cr}\mspace{14mu} {release}} - {{{spontaneous}\mspace{14mu}}^{51}{Cr}\mspace{14mu} {release}}}{{{{Total}{\; \mspace{11mu}}}^{51}{Cr}\mspace{14mu} {release}} - {{{spontaneous}\mspace{14mu}}^{51}{Cr}\mspace{14mu} {release}}}$

19-B: Antibody-Dependent Macrophage-Mediated Cytotoxicity Assay

To determine whether the anti-PSCA mAbs sensitize tumor cells to ADMMC,the following assay is performed. Peritoneal macrophages are activatedby intraperitoneal injection of SCID mice with Brewer's thioglycollatemedium as described by Larson et al., 1988, Int. J. Cancer 42: 877-882.After four days, cells are collected by intraperitoneal lavage, and thepercent of activated macrophages determined by Mac-1 staining. For theassay, the activated macrophages are co-cultured with ³H-thymidinelabeled LAPC-9, LNCaP-PSCA, and LNCaP target cells for 48 hours in thepresence of either anti-PSCA mAbs or control mouse IgG. At the end ofthe assay, supernatants are harvested from the wells and killing isdetermined by the amount of ³H-thymidine released as described above for⁵¹Cr release.

19-C: Complement-Mediated Tumor Cell Lysis Assay

Destruction of tumor targets by complement-dependent lysis may beperformed according to the method described by Huang et al., 1995,Cancer Res. 55: 610-616. For example, LAPC-9, LNCaP-PSCA, and LNCaPcells are labeled with ⁵¹Cr and then incubated on ice for 30′ witheither anti-PSCA mAbs or a mouse IgG control. After washing to removeunbound antibody, the cells are incubated with rabbit complement at 37°C. for 2 hr, and cell lysis measured by ⁵¹Cr-release into thesupernatant. The percent cell lysis will be determined as describedabove.

19-D: Cell Proliferation Assay

The effect of anti-PSCA mAbs on cell proliferation may be determined byan MTT assay. Briefly, LNCaP-PSCA or LNCaP cells are cultured for 72 hrwith varying amounts of either anti-PSCA mAbs or mouse IgG as a control.At the end of the incubation period, the cells are washed and incubatedin a solution of MTT for 4 hr. Proliferation is indicated bydehydrogenase mediated conversion of the MTT solution to a purple colorand measured at a wavelength of 570 nm.

19-E: Assay for Colony Formation in Soft Agar

Colony formation in the presence of anti-PSCA mAbs may be measured bygrowth of cells in soft agar. Briefly, 1×10⁴ LNCaP-PSCA or LNCaP cellsare plated in medium containing Nobel agar. A dilution series ofanti-PSCA mAbs is then added to plates in duplicate to determine theeffect on colony growth. Mouse IgG is used as a control. Macroscopiccolonies are counted after 14-21 days in culture.

Example 20 PSCA Capture ELISA

A PSCA capture ELISA was developed in order to measure PSCA levels inserum prior to treatment with anti-PSCA mAbs and provides informationuseful in determining the therapeutic dosage regimen. The assay may alsobe useful in monitoring patient response to the therapy.

A schematic representation of the assay format is shown in FIG. 50B.Briefly, affinity purified anti-PSCA peptide sheep polyclonal antibody(directed against amino acids 67-81 of the PSCA protein) and anti-PSCAmonoclonal antibody 1G8 are used as capture antibodies and are coatedmicrotiter wells. After coating, incubation with a dilution series oftest antigen is conducted in order to generate a standard curve. Patientserum is added to the wells and incubated at room temperature. Afterincubation, unbound antibody is washed with PBS. Anti-PSCA monoclonalantibodies 2A2, 3C5 and 4A10 (IgG2a isotype), which recognize differentepitopes on the PSCA protein, are used as detection antibodies, and areadded to the wells, incubated, and the wells washed to remove unboundantibody. The captured reaction is then visualized by the addition of ananti-mouse Ig2a-horseradish peroxidase-conjugated secondary antibodyfollowed by development with 3,3′ 5,5′ tetramethylbenzidine basesubstrate and OD determinations taken.

A schematic representation of the standardization and control antigensare shown in FIG. 50A. Briefly, a GST-fusion protein encoding aminoacids 18-98 of PSCA is used for generating a standard curve forquantification of unknown samples. A secreted recombinant mammalianexpressed form of PSCA is used for quality control of the ELISA assay.This protein contains an Ig leader sequence to direct secretion of therecombinant protein and MYC and 6×HIS (SEQ ID NO:16) epitope tags foraffinity purification.

Quantification of recombinant PSCA secreted from 293T cells engineeredto express and secrete PSCA is shown in FIG. 51A-B.

Example 21 Sequence of PSCA mAb Genes

The nucleotide sequences of the genes encoding the heavy chain variableregions of murine monoclonal antibodies 1G8, 4A10 and 2H9 weredetermined using the methods described in Coloma et al., 1992, JImmunol. Methods 153: 89-104. Primers for heavy chain variable regionsequencing of mAbs 1G8 and 4A10 were as follows:

HLEAD.1: (SEQ ID NO: 19)ggc gat atc cac cat ggR atg Sag ctg Kgt Mat Sct ctt  CH3′:(SEQ ID NO: 20) agg gaa ttc aYc tcc aca cac agg RRc cag tgg ata gac 

Primers for heavy chain variable region sequencing of mAb 2H9 were asfollows:

HLEAD.2: (SEQ ID NO: 21)ggg gat atc cac cat gRa ctt cgg gYt gag ctK ggt ttt  CH3′: (SEQ ID NO: 20) agg gaa ttc aYc tcc aca cac agg RRc cag tgg ata gac 

Total RNA was isolated from 1G8, 2H9, and 4A10 hybridoma cells using theTrizol Reagent (Gibco-BRL cat#15596). First strand synthesis reactionson 5 μg of RNA were generated using the Gibco-BRL Superscript II reversetranscriptase reaction according to manufacturers protocols and CH3′.Two μl of the 30 μl first strand reaction was used in the PCR to amplifythe variable regions.

First strand cDNA was synthesized from hybridoma RNA using a primer fromthe constant region of the heavy chain (CH3′). The variable region wasamplified using CH3′ and a primer designed to the leader sequence(HLEAD.1 and HLEAD.2). The resulting PCR product is sequenced and thecomplementarity determining regions (CDRs) are determined using theKabat rules. The sequences are shown in FIGS. 58, 59 and 60. An aminoacid alignment of the CDRs of these three mAbs is shown in FIG. 61.

Example 22 PSCA mAb Binding Affinity

The affinity of PSCA monoclonal antibody 1G8 (described above) wasdetermined using BIAcore™ instrumentation (Uppsala, Sweden), which usessurface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect.23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) tomonitor biomolecular interactions in real time. On the basis of generalprocedures outlined by the manufacturer (Pharmacia), kinetic analysis ofthe antibody was performed using a recombinant PSCA immobilized onto thesensor surface at a low density (30 RU). Recombinant PSCA was generatedas follows. 293T cells transiently transfected or 293 cells stablyexpressing a CMV-driven expression vector encoding PSCA with aC-terminal 6×His (SEQ ID NO:16) and MYC tag (pcDNA3.1/mycHIS,Invitrogen) served as source of secreted soluble PSCA protein forpurification. The HIS-tagged PSCA protein was purified over a nickelcolumn using standard techniques. The association and dissociation rateswere determined using the software provided by the manufacturer. Theresults, tabulated below (Table 5), show that 1G8 has a 1 nanomolarK_(D), indicating a strong affinity for the PSCA antigen.

TABLE 5 BIOCORE AFFINITY DETERMINATION OF PSCA mAb 1G8 mAb in solutionk_(a) (M⁻¹s⁻¹) k_(d) (M⁻s⁻¹) K_(D) (nM) 1.68 × 10⁵ 1.69 × 10⁴ 1.0

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Example 23 Immunohistochemical Analysis

This example describes immunohistochemical (IHC) analysis of variousformalin-fixed, paraffin-embedded tissues with the seven anti-PSCA mAbsdescribed supra.

Materials and Methods

Each of the seven anti-PSCA monoclonal antibodies was tested against:(1) a cell pellet consisting of LNCaP overexpressing PSCA, LNCaPparental, and 293T cells; (2) LAPC9AD xenograft; and (3) benignprostatic hyperplasia (BPH), prostate carcinoma, and normal prostatetissues. All tissue staining was performed by QualTek MolecularLaboratories, Inc (Santa Barbara, Calif.). Tissue blocks were sectionedat 4 microns and placed onto positively charged Capillary Gap microscopeslides (Ventana Medical Systems, Inc., Tucson, Ariz.). After dewaxing inxylene, followed by hydration through alcohol series, tissue sectionswere pretreated in a steamer for 20 minutes in the presence of sodiumcitrate (10 mM, pH 6.0) in order to optimize antibody reactivity.

After cooling for 5 minutes, the slides were immunostained using anABC-peroxidase technique. Briefly, slides were incubated in blockingserum (normal goat) for 5 minutes, followed by 2 μg/ml anti-PSCAmonoclonal primary antibody or 2 μg/ml mouse IgG for the negativecontrol (25 minutes), biotinylated secondary antibody-goat-anti-mouseIgG (25 min) and avidin-biotin complex (ABC) conjugated to peroxidaseenzyme, Vector

Labs, Burlingame, Calif. (25 minutes). Between the incubation steps,sections were rinsed in buffer. DAB—Diaminobenzidine chromogen (QualTekMolecular Labs) was used to develop the reaction—yielding a brownprecipitate. Slides were subsequently counterstained with hematoxylinand then coverslipped. Staining was performed on a TechMate 1000automated staining instrument (Ventana Medical Systems, Inc., Tucson,Ariz.) at room temperature.

Results

FIG. 52 shows the IHC results for the anti-PSCA monoclonal antibody 3C5in the cell pellet, LAPC9AD xenograft, a BPH sample, and a prostatecarcinoma tissue (left panel). The cell pellet mix contains three celltypes of which only one, the LNCaP-PSCA cells, are expected to stainwith anti-PSCA monoclonal. As expected, 3C5 stains stronglyapproximately ⅓ of the cells. This staining conveniently shows positiveand negatively staining cell types on the same slide. The LAPC9ADxenograft is very strongly stained with 3C5 antibodies. Basal andepithelia cells in the ducts of the BPH tissue stain well but the basalcells are especially prominent. Finally, the prostate carcinoma tissueshows strong staining in the neoplastic ducts. The panel on the rightrepresents the background control consisting of pre-immune mouse serum.No background staining was detectable in any of the samples evaluated.

Example 24

Inhibition of established LAPC-9 tumor growth and prolonged survivalfollowing anti-PSCA antibody treatment. The LAPC-9 xenograft wasgenerated from a bone tumor biopsy of a patient with hormone-refractorymetastatic prostate cancer.

The following examples demonstrate that anti-PSCA monoclonal antibodies,1G8 and 3C5, inhibit the growth of established orthotopic, LAPC-9prostate tumors, in SCID mice and significantly prolonged theirsurvival. The growth of the tumors was monitored by tracking the levelof serum PSA.

Orthotopic Injections

A single cell suspension of LAPC-9 tumors was prepared according to themethods described in Example 18-A. The cell suspension was mixed, at a1:1 dilution, with Matrigel. The cell suspension was kept on ice priorto the orthotopic injections. The orthotopic injections were performedon male IcR-SCID mice, under anesthesia, using ketamine/xylazine. Theanesthetized mice were shaved in the lower abdomen, a 5-8 mm incisionwas made just above the penis to expose the abdominal wall and muscles.An incision was made through the abdominal muscles to expose the bladderand seminal vescicles, which were then delivered through the incision toexpose the dorsal prostate. The LAPC-9 cell suspension was injected intoeach dorsal lobe using a 500 μl Hamilton syringe. Each lobe was injectedwith 10 μl of cell suspension corresponding to about 5×10⁵ cells. Afterthe injections, both incisions were closed using a running suture andthe mice were kept under a heat lamp to recover. After the injections,the serum level of PSA was monitored. The mice were treated with adifferent regimen of 1G8 or 3C5 antibody, depending on the serum levelof PSA. After the antibody treatments, the mice were kept alive, todetermine the PSA levels as a measure of the tumor growth, and todetermine the survival of the mice in each treatment group.

Monitoring the Serum Levels of PSA:

The level of serum PSA was used to track the tumor size. The mice werebled on an approximate weekly basis, to monitor the levels of serum PSA,which were measured using a commercially-available PSA kit (AmericanQualex, San Clemente, Calif.). The mice were segregated into twotreatment groups, based on the levels of serum PSA. For example, thegroups included: low levels of PSA (e.g., 2-3 ng/ml; FIG. 66 A); andmoderate levels of PSA (e.g., 64-78 ng/ml; FIG. 66 B). The serum PSAlevels were monitored until the tumors were visible through the abdomen.

The serum PSA levels were monitored on days +9, +15, +22, +30, +37, +44,and +51, relative to the day of injection of the tumor cells (FIG. 66A). Similarly, the serum PSA levels were monitored on days +13, +21,+27, and +34 (FIG. 66 B), on days +9, +16, +22, +29, and +36 (FIG. 68A), and on days +7, +14, +21, and +28 (FIG. 68 B).

Treatment with 1G8:

Orthotopic, tumor-bearing mice were established according to the methodsdescribed above. Two groups of animals, exhibiting (i) low levels ofserum PSA (2-3 ng/ml), or (ii) moderate levels of serum PSA (64-78ng/ml) were selected for treatment.

The mice having low levels of PSA (e.g., 2-3 ng/ml) were treated withintraperitoneal injection of 2 mg of 1G8 antibody, three times per weekfor one week, followed by 1 mg of 1G8 three times per week for the nexttwo weeks, followed by 1 mg of 1G8 once per week for the next threeweeks (as indicated by the arrows in FIG. 66 A).

The mice having moderate levels of PSA (e.g., 64-78 ng/ml) were treatedwith intraperitoneal injection of 1 mg of 1G8 antibody, three times perweek for three consecutive weeks, followed by a single injection of 1 mgof 1G8 the following week (as indicated by the arrows in FIG. 66 B).

The control mice, having low or moderate levels of PSA, were treatedwith about 0.5 to 0.8 ml of phosphate buffer solution (Gibco) (FIGS. 66A and B).

Treatment with 3C5:

Similar treatments were performed on orthotopic tumor-bearing mice,using the 3C5 antibody. In the first experiment, 1 mg of 3C5 antibodywas administered intraperitoneally three times per week for threeconsecutive weeks, followed by a single injection of 1 mg of 3C5 (FIG.68 A). In the second experiment, 2 mg of 3C5 was administered threetimes per week for the first week, followed by 1 mg three times per weekfor the next two weeks (FIG. 68 B). The injections were administered onthe days indicated by the arrows in FIGS. 68 A and B.

Results—Treatment with 1G8 Results in Inhibition of Tumor Growth andIncreased Survival:

The serum PSA levels of the tumor-bearing mice were used to track thegrowth of the tumors, since the serum PSA levels correlated well withthe tumor size. The mice bearing LAPC-9 AD tumors, treated with theanti-PSCA monoclonal antibody, 1G8, exhibited a reduction in the rate ofincrease in serum PSA levels (FIGS. 66 A and B). This result indicatesthat 1G8 inhibits growth of tumors expressing PSCA.

In FIG. 66 A, each data point represents the mean PSA level for micereceiving PBS (n=6) or 1G8 (n=6). The mice were bled on the daysindicated on the X-axis for PSA determinations.

As shown in FIG. 66 B, the mice were bled on the days indicated on theX-axis for PSA determinations. Each data point represents the mean PSAlevel for mice receiving PBS (n=4) or 1G8 (n=5). In FIG. 67A-B, 4 micein the PBS-treated group and 5 mice in the 1G8-treated group.

Additionally, the mice having lower serum PSA levels that were treatedwith the 1G8 antibody exhibited a reduced rate of increase in the levelof serum PSA, compared to the mice having higher serum PSA levels thatwere treated with the 1G8 antibody (e.g., compare the data described by(▪) in FIGS. 66 A and B). This result suggests that the 1G8 antibody wasmore effective at reducing tumor growth, when there was a smaller tumorburden, under the administration protocol of this study.

The affect of the 1G8 treatment on survival of the tumor-bearing micewas also monitored. In general, the mice treated with only PBS began todie within 5-6 weeks post-injection, due to local tumor growth andmetastasis. In contrast, the mice treated with 1G8 antibody exhibited aprolonged life. For example, the effect on survival was more dramatic inthe mice having low serum PSA levels (FIG. 67 A), where the mediansurvival time in the PBS-treated mice was 56.5 days (range 42-71 days)and the median survival time in the 1G8-treated mice was 89 days (range77-101). In the mice having moderate serum PSA levels (FIG. 67 B) themedian survival time in PBS-treated mice was 40 days (range 32-48 days)compared to a median survival time of 78.5 days (range 52-105 days) inthe 1G8-treated mice. This indicates an increase of median survival timeof 38.5 days in 1G8-treated mice.

The inhibition of tumor growth correlated with prolonged life.Collectively, these results demonstrate that 1G8 treatment inhibitedtumor growth and increased the survival time of orthotopic tumor-bearingmice. Thus, these results suggest that treatment with 1G8 increasedsurvival time, by inhibiting tumor growth.

Treatment with 1G8 effectively inhibited tumor growth on smallerorthotopic tumors. Thus, 1G8 may represent an effective therapeutictreatment for minimal residual disease, in recurrent local disease,after primary treatment and in metastatic tumor formation.

The results of mice treated with the 3C5 antibody are similar to thedata obtained from mice treated with the 1G8 antibody. The mice bearingLAPC-9 AD tumors, treated with the anti-PSCA monoclonal antibody, 3C5,exhibited a decrease in serum PSA levels. This result indicates that 3C5inhibits growth of tumors expressing PSCA.

Two separate experiments were conducted to evaluate the effect of 3C5treatment. The mice treated with 3C5 antibody exhibited a lower rate ofincrease in the level of serum PSA, compared to the mice treated withphosphate buffer solution (FIGS. 68 A and B). This result suggests thatthe 3C5 antibody inhibited tumor growth.

In FIG. 68 A, each data point represents the mean PSA level for micereceiving PBS (n=4) or 3C5 (n=5). In FIG. 68 B, each data pointrepresents the mean PSA level for mice receiving PBS (n=6) or 3C5 (n=6).

The mice treated with 3C5 antibody exhibited prolonged life (FIG. 69 A),compared to the mice treated with PBS (FIG. 69 B). In the firstexperiment, the median survival time of the PBS-treated mice was 52 days(range 45-59 days), compared to more than 92 days (range 59 to +125days) in the 3C5-treated group (FIG. 69 A) (one mouse is still alive).In the second experiment, the median survival time in PBS-treated micewas 43 days (range 29-57), compared to 57.5 days in the 3C5-treated mice(range 33-82 days) (FIG. 69 B).

The inhibition of tumor growth correlated with prolonged life.Collectively, these results demonstrate that 3C5 treatment inhibitedtumor growth and thereby increased the survival time of orthotopictumor-bearing mice. Thus, these results indicate that treatment with 3C5increased survival time, by inhibiting tumor growth.

Example 25

Inhibition of established PC3-PSCA tumor growth and prolonged survivalfollowing anti-PSCA antibody treatment alone or in combination withdoxorubicin.

The following examples demonstrate that 1G8, an anti-PSCA monoclonalantibody, inhibited the growth of established subcutaneous, PC3-PSCAprostate tumors (AI), growing in SCID mice. Additionally, 1G8, whenadministered alone or in combination with doxorubicin, inhibited thegrowth of prostate tumors. Furthermore, treatment with 1G8 prolonged thesurvival of these mice, when administered alone or in combination withdoxorubicin. Treatment with 1G8 and doxorubicin appears to result in asynergistic inhibitory effect on tumor growth and survival.

PSCA-Expressing PC3 Cells:

PC3-PSCA cells were derived by retroviral gene transfer. Briefly, PSCAcDNA was inserted into the retroviral vector pSR-α (Muller, et al., 1991Molec. Cell. Biol. 11:1785-1792)). Amphotropic retrovirus was generatedby co-transfection of 293T cells with pSR-a containing PSCA and aretroviral helper plasmid containing the amphotropic envelope protein.PC3 cells were subsequently infected with the PSCA containingamphotropic retrovirus, and 48 hours after infection the cells wereselected by growth in medium containing the neomycin analogue G418.After 2-3 weeks of selection and expansion, a Western blot was performedto confirm that the PC3-PSCA cells express PSCA protein. Parental PC3 orPC3 cells infected with an empty vector that did not contain PSCA wereboth negative for PSCA protein expression.

Subcutaneous Injections:

PC3-PSCA cells were grown in T-150 flasks in DMEM+10% FBS prior to theinjections. The cells were grown to log phase, harvested, washed,counted, then mixed with Matrigel at a 1:1 dilution, and kept on ice.For injection, male IcR-SCID mice were shaved on their flanks, and eachmouse received a single subcutaneous injection of about 1×10⁶ cells in avolume of 100 μl on the right flank. The growth of PC3-PSCA tumors wasfollowed by caliper measurements of the growing tumors. The mice wereselected for treatment when the tumor reached the size of 100-200 mm³,at approximately 9 days after the subcutaneous injection. The tumor sizewas measured at days +9, +15, +23, +29, +36, and +43 post injection.

Treatment with PBS:

The control mice were treated with about 0.5 to 0.8 ml of phosphatebuffer solution (Gibco) (FIGS. 66 A and B).

Treatment with 1G8:

The mice treated with 1G8, were administered 1 mg of 1G8, three timesper week for three consecutive weeks.

Treatment with Doxorubicin:

An LD₅₀ experiment was performed to determine the maximum tolerable doseof doxorubicin. Doxorubicin (Sigma) and was resuspended in sterile PBS.Doxorubicin was administered by intraperitoneal injection, at thefollowing doses: 50 μg, 25 μg, 12.5 μg, and 6.75 μg. At the highestdose, 50 μg, all the mice died within 2 weeks. At the lower dose ranges,the mice survived for more than 4 weeks. The maximal tolerable dose was25 μg.

The mice treated with only doxorubicin, were administered 25 μg, onceweekly for three consecutive weeks.

Treatment with 1G8 and Doxorubicin:

The mice treated with 1G8 and doxorubicin, were administered 1 mg of 1G8three times per week for three consecutive weeks (FIG. 70; 1G8=arrows),and were administered 25 μg of doxorubicin once weekly for threeconsecutive weeks (FIG. 70; doxorubicin=()).

Results—Treatment with 1G8 Alone or in Combination with DoxorubicinResults in Inhibition of Tumor Growth:

The mice bearing PC3-PSCA tumors, treated with anti-PSCA monoclonalantibody, 1G8, alone or in combination with doxorubicin, exhibited adecrease in tumor growth compared to mice treated with phosphate buffersolution or doxorubicin alone (FIG. 70). These results indicate that 1G8inhibits the growth of tumors expressing PSCA. These results alsosuggest that the combination of 1G8 and doxorubicin act synergisticallyto inhibit tumor growth.

Each data point represents the mean tumor volume for mice receiving PBS(n=5), doxorubicin (n=6), 1G8 (n=6), or 1G8+doxorubicin (n=6).

The mice treated with doxorubicin exhibited a slightly lower tumorgrowth rate, compared to mice treated with PBS (e.g., 4% growthinhibition at day 43). In contrast, mice treated with 1G8 antibody aloneexhibited a greater reduction in tumor growth rate, compared to the micetreated with PBS (e.g., 20% growth inhibition at day 43). The micetreated with the combination of 1G8 and doxorubicin exhibited a slightlygreater reduction in tumor growth rate, compared to the mice treatedwith PBS (e.g., 36% growth inhibition at day 43).

Treatment with 1G8 alone, in this subcutaneous model using PC3-PSCAxenografts, effectively inhibited tumor growth on establishedandrogen-independent tumors (e.g., PC3 cells are hormone refractory).Furthermore, the combination of 1G8 and doxorubicin showed augmentedtumor growth inhibitory effects. Thus, treatment with the combination of1G8 and doxorubicin represents an effective therapeutic treatment forprostate cancer patients with metastatic disease who have failed hormoneablation therapy.

Example 26 Anti-PSCA Monoclonal Antibodies Circulate and TargetPSCA-Expressing Tumors

In one study, SCID mice bearing established, subcutaneous, LAPC-9 ADtumors, described in Example 18 C4 above, were treated with eithercontrol mouse IgG or 3C5 anti-PSCA mAb as described. On day 34, 6 daysafter the last antibody treatment, the mice were sacrificed and thetumors were explanted. In a different study, tumor-bearing SCID micewere treated with control mouse IgG or 1G8. The presence of antibody inthe tumor samples from both studies was detected by either Western blotanalysis or immunohistochemistry (IHC).

Immunohistochemistry:

Explanted tumors were fixed in formalin and embedded in paraffin forimmunohistochemical analysis (performed by QualTek Molecular Labs, SantaBarbara, Calif.). The paraffin blocks were sliced, the slices were fixedon slides, and the slides were probed with biotinylated goat anti-mouseIgG followed by an avidin-biotin complex (ABC) conjugated to peroxidaseenzyme (Vector Labs, Burlingame, Calif.). DAB (diaminobenzidine)chromogen was used to develop the reaction which yielded a brownprecipitate. Slides were subsequently counterstained with hematoxylinand then coverslipped. Staining was performed on a TechMate 1000automated staining instrument (Ventana Medical Systems, Inc., Tucson,Ariz.) at room temperature (FIG. 71).

Results—Immunohistochemistry:

FIG. 71 demonstrates that the 3C5 antibody localized in the LAPC-9 ADtumors from the 3C5-treated mice, but not with control IgG-treated mice.Additionally, antibody could be detected throughout the tumor.

Western Blotting:

Explanted tumors from 3 mice in the IgG-treated group and the3C5-treated group, (e.g., the mice as described in Example 18 C4 above),were lysed in boiling SDS sample buffer. The cell lysates wereelectrophoresed in SDS-PAGE gels, transferred to nitrocellulose filters,probed with goat anti-mouse IgG-HRP antibodies (Southern Biotech,Birmingham, Ala.), and visualized by enhanced chemiluminescence. Themouse IgG control antibody and 3C5 were also run on the gel as controls(FIG. 72).

In a similar experiment, LAPC-9 AD subcutaneous tumor-bearing mice weretreated with either control mouse IgG or the 1G8 anti-PSCA mAb. On day30, which was 7 days after the last antibody treatment, the mice weresacrificed and tumors were explanted. Western blot analysis wasperformed on explanted tumors from 3 mice in each group. The explantedtumors were lysed in boiling SDS sample buffer, the cell lysates wereelectrophoresed in SDS-PAGE gels, transferred to nitrocellulose, probedwith goat anti-mouse IgG-HRP antibodies (Southern Biotech, Birmingham,Ala.), and visualized by enhanced chemiluminescence. The mouse IgGcontrol antibody and 1G8 were also run on the gel as controls (FIG. 73).

Results—Western Blotting:

FIG. 72 demonstrates that IgG heavy and light chains were readilydetected in tumor lysates from the 3C5 treated mice, but not in themouse IgG control treated mice.

FIG. 73 demonstrates that IgG heavy and light chains were readilydetected in tumor lysates from the 1G8 treated mice, but not in themouse IgG control treated mice.

These results demonstrate that anti-PSCA mAbs, such as 3C5 and 1G8, cancirculate and target a PSCA-expressing tumor, after administration totumor-bearing mice. Furthermore the antibody localization is specificsince control mouse IgG, which does not recognize PSCA, is either absentfrom the tumors, or present at very low levels when compared to tumorsfrom anti-PSCA treated mice. These results suggest that anti-PSCA mAbshave the potential to circulate through the body and localize to primaryand metastatic, PSCA-expressing tumors in cancer patients. Furthermore,conjugated anti-PSCA mAbs may be capable of effectively destroyingPSCA-expressing tumors for local, locally recurring and metastaticdisease.

What is claimed is:
 1. An immunotherapy method comprising administeringto a subject having a disease associated with the expression of ProstateStem Cell Antigen (PSCA) a therapeutically effective amount of anantibody, wherein the antibody is a monoclonal antibody designated 1G8(American Tissue Type Culture Collection (ATCC) No. HB-12612) or afragment thereof, a humanized form of the 1G8 monoclonal antibody, animmunoconjugate comprising the 1G8 monoclonal antibody, or a fusionprotein comprising the fragment of the 1G8 monoclonal antibody.
 2. Themethod of claim 1, wherein the disease is associated with theoverexpression of PSCA.
 3. The method of claim 1, wherein the disease isa cancer.
 4. The method of claim 3, wherein the cancer is bladdercancer, prostate cancer, metastasis of prostate cancer, or pancreaticcancer.
 5. The method of claim 1, wherein the antibody is a 1G8monoclonal antibody deposited as ATCC No. HB-12612.
 6. The method ofclaim 1, wherein the fragment comprises a Fab, F(ab′)₂, or Fv.
 7. Themethod of claim 1, wherein the antibody is a humanized form of the 1G8monoclonal antibody.
 8. The method of claim 1, wherein the antibody isan immunoconjugate comprising the 1G8 monoclonal antibody linked to atherapeutic agent.
 9. The method of claim 8, wherein the therapeuticagent is an anti-tumor drug, a cytotoxic agent, a radioactive agent, acytokine, a second antibody, or an enzyme.
 10. The method of claim 9,wherein the therapeutic agent is a cytotoxic agent.
 11. The method ofclaim 9, wherein the therapeutic agent is a radioactive isotope.
 12. Themethod of claim 1, wherein the antibody is a fusion protein comprisingthe fragment of the 1G8 monoclonal antibody.
 13. The method of claim 1,wherein the method further comprises administering a chemotherapeuticdrug to the subject.
 14. The method of claim 1, wherein the methodfurther comprises administering radiation therapy to the subject.